Cationic lipids containing silicon

ABSTRACT

Certain embodiments of the invention provide a cationic lipid of formula (I): wherein R 1 , R 2 , R 3  and R 4  are defined as described herein, as well as methods of making these lipids. Certain embodiments of the invention also provide nucleic acid-lipid particles comprising a cationic lipid of formula (I), methods of making the lipid particles, and methods of delivering and/or administering the lipid particles.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This patent application claims the benefit of priority of U.S.application Ser. No. 62/758,108, filed Nov. 9, 2018, which applicationis herein incorporated by reference.

BACKGROUND

Cationic lipid containing nanoparticles have been used for the deliveryof a variety of active agents and therapeutic agents. Currently thedifficulty and cost associated with the preparation of the cationiclipid component of these nanoparticles limit their attractiveness forcommercial development as delivery vehicles. Accordingly, there is aneed for additional cationic lipids that can be incorporated into lipidnanoparticles. In particular, there is a need for cationic lipids thatcan be prepared using less expensive and more efficient processes. Thereis also a need for cationic lipids that are relatively morebiodegradable, such that the related lipid nanoparticles better degradefollowing delivery of an active agent or a therapeutic agent.

SUMMARY

The invention provides cationic lipids that can be prepared using lessexpensive and more efficient processes. Additionally, the cationiclipids are effective for delivering an active agent or therapeuticagent, such as a nucleic acid, when incorporated into lipidnanoparticles. The cationic lipids are also biodegradable, such thatassociated lipid nanoparticles better degrade following delivery of anactive agent or a therapeutic agent.

In one embodiment, the invention provides a cationic lipid of formula(I):

wherein:

R¹ is a C₂-C₃₀ hydrocarbyl;

R² is a C₂-C₃₀ hydrocarbyl;

R³ is a C₂-C₃₀ hydrocarbyl;

X is a divalent C₂-C₈ alkyl;

R⁴ is NR^(a)R^(b); and

each R^(a) and R^(b) is independently selected from the group consistingof methyl, ethyl, propyl, cyclopropyl, and butyl, which methyl, ethyl,propyl, cyclopropyl, and butyl is optionally substituted with hydroxy;or R^(a) and R^(b) taken with the nitrogen to which they are attachedform an aziridine, azetidine, proline, piperidine, piperazine, ormorpholine ring, which ring is optionally substituted with hydroxyl orwith C₁-C₆ alkyl that is optionally substituted with hydroxy.

The invention also provides a composition comprising 1) a compound offormula (I), and 2) an active agent or a therapeutic agent.

The invention also provides a lipid nanoparticle comprising a compoundof formula (I).

The invention also provides a lipid nanoparticle comprising: (a) one ormore active agents or therapeutic agents; (b) a compound of formula (I);(c) a non-cationic lipid; and (d) a conjugated lipid. Typically, the oneor more agents are encapsulated within the lipid nanoparticle

The invention also provides a pharmaceutical composition comprising, 1)a compound of formula (I), and 2) one or more active agents ortherapeutic agents.

The invention also provides a pharmaceutical composition comprising alipid nanoparticle of the invention and a pharmaceutically acceptablecarrier.

The invention also provides a method for delivering an active agent or atherapeutic agent (e.g. a nucleic acid) to an animal, comprisingadministering a nanoparticle of the invention to the animal.

The invention also provides a method for introducing a nucleic acid intoa cell, the method comprising, contacting the cell in vivo, ex vivo orin vitro with a nucleic acid-lipid particle described herein.

The invention also provides a method for treating a disease or disorderin an animal, comprising administering a therapeutically effectiveamount of a nucleic acid-lipid particle described herein to the animal.

The invention also provides processes and intermediates disclosed hereinthat are useful for making the compounds, formulations, or nanoparticlesof the invention. For example, in one embodiment, the invention providesa method for preparing a compound of formula (I):

wherein R⁴ is NR^(a)R^(b) as described herein, comprising reacting acorresponding compound of formula (Ia):

wherein R^(4a) is bromo or chloro, with an amine of formula HNR^(a)R^(b)to provide the compound of formula (I).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence”refers to single-stranded RNA (e.g., mature miRNA) or double-strandedRNA (i.e., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that iscapable of reducing or inhibiting the expression of a target gene orsequence (e.g., by mediating the degradation or inhibiting thetranslation of mRNAs which are complementary to the interfering RNAsequence) when the interfering RNA is in the same cell as the targetgene or sequence. Interfering RNA thus refers to the single-stranded RNAthat is complementary to a target mRNA sequence or to thedouble-stranded RNA formed by two complementary strands or by a single,self-complementary strand. Interfering RNA may have substantial orcomplete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule.

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequencethat does not have 100% complementarity to its target sequence. Aninterfering RNA may have at least one, two, three, four, five, six, ormore mismatch regions. The mismatch regions may be contiguous or may beseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.The mismatch motifs or regions may comprise a single nucleotide or maycomprise two, three, four, five, or more nucleotides.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a nucleic acid (e.g., an interferingRNA or mRNA) is an amount sufficient to produce the desired effect,e.g., an inhibition of expression of a target sequence in comparison tothe normal expression level detected in the absence of an interferingRNA; or mRNA-directed expression of an amount of a protein that causes adesirable biological effect in the organism within which the protein isexpressed. Inhibition of expression of a target gene or target sequenceis achieved when the value obtained with an interfering RNA relative tothe control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other embodiments, theexpressed protein is an active form of a protein that is normallyexpressed in a cell type within the body, and the therapeuticallyeffective amount of the mRNA is an amount that produces an amount of theencoded protein that is at least 50% (e.g., at least 60%, or at least70%, or at least 80%, or at least 90%) of the amount of the protein thatis normally expressed in the cell type of a healthy individual. Suitableassays for measuring expression of a target gene or target sequenceinclude, e.g., examination of protein or RNA levels using techniquesknown to those of skill in the art such as dot blots, northern blots, insitu hybridization, ELISA, immunoprecipitation, enzyme function, as wellas phenotypic assays known to those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA). The amount of decrease of an immune response by amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. Adetectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morelower than the immune response detected in the presence of theunmodified interfering RNA. A decrease in the immune response tointerfering RNA is typically measured by a decrease in cytokineproduction (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by a responder cellin vitro or a decrease in cytokine production in the sera of a mammaliansubject after administration of the interfering RNA.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an mRNA is intended to mean a detectable decrease of animmune response to a given mRNA (e.g., a modified mRNA). The amount ofdecrease of an immune response by a modified mRNA may be determinedrelative to the level of an immune response in the presence of anunmodified mRNA. A detectable decrease can be about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more lower than the immune response detected in thepresence of the unmodified mRNA. A decrease in the immune response tomRNA is typically measured by a decrease in cytokine production (e.g.,IFNγ, IFNα, TNFα, IL-6, or IL-12) by a responder cell in vitro or adecrease in cytokine production in the sera of a mammalian subject afteradministration of the mRNA.

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β,IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, andcombinations thereof.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Preferably, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology, Ausubelet al., eds. (1995 supplement)).

A preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.,215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA and RNA. DNA may be in the formof, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCRproduct, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, chromosomal DNA, or derivatives andcombinations of these groups. RNA may be in the form of siRNA,asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA,rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinationsthereof. Nucleic acids include nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

As used herein, the term “LNP” refers to a lipid-nucleic acid particleor a nucleic acid-lipid particle (e.g., a stable nucleic acid-lipidparticle). A LNP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and a conjugated lipid thatprevents aggregation of the particle), and a nucleic acid, wherein thenucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, shorthairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or a plasmid,including plasmids from which an interfering RNA or mRNA is transcribed)is encapsulated within the lipid. In one embodiment, the nucleic acid isat least 50% encapsulated in the lipid; in one embodiment, the nucleicacid is at least 75% encapsulated in the lipid; in one embodiment, thenucleic acid is at least 90% encapsulated in the lipid; and in oneembodiment, the nucleic acid is completely encapsulated in the lipid.LNPs typically contain a cationic lipid, a non-cationic lipid, and alipid conjugate (e.g., a PEG-lipid conjugate). LNP are extremely usefulfor systemic applications, as they can exhibit extended circulationlifetimes following intravenous (i.v.) injection, they can accumulate atdistal sites (e.g., sites physically separated from the administrationsite), and they can mediate expression of the transfected gene orsilencing of target gene expression at these distal sites.

The lipid particles of the invention (e.g., LNP) typically have a meandiameter of from about 40 nm to about 150 nm, from about 50 nm to about150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110nm, or from about 70 to about 90 nm, and are substantially non-toxic. Inaddition, nucleic acids, when present in the lipid particles of theinvention, are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and20070042031, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA or mRNA), with full encapsulation, partialencapsulation, or both. In a preferred embodiment, the nucleic acid isfully encapsulated in the lipid particle (e.g., to form an SPLP, pSPLP,LNP, or other nucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, polyamide oligomers (e.g., ATTA-lipid conjugates),PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEGcoupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled tophosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S.Pat. No. 5,885,613, the disclosure of which is herein incorporated byreference in its entirety for all purposes), cationic PEG lipids, andmixtures thereof. PEG can be conjugated directly to the lipid or may belinked to the lipid via a linker moiety. Any linker moiety suitable forcoupling the PEG to a lipid can be used including, e.g., non-estercontaining linker moieties and ester-containing linker moieties. Inpreferred embodiments, non-ester containing linker moieties are used.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “cationic lipid” refers to a compound of Formula (I) asdescribed herein.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa LNP, to fuse with the membranes of a cell. The membranes can be eitherthe plasma membrane or membranes surrounding organelles, e.g., endosome,nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as LNPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent ortherapeutic agent, such as an interfering RNA or mRNA, within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. To obtain broad biodistribution generally requires ablood lifetime such that the agent is not rapidly degraded or cleared(such as by first pass organs (liver, lung, etc.) or by rapid,nonspecific cell binding) before reaching a disease site distal to thesite of administration. Systemic delivery of lipid particles can be byany means known in the art including, for example, intravenous,subcutaneous, and intraperitoneal. In a preferred embodiment, systemicdelivery of lipid particles is by intravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentor therapeutic agent, such as an interfering RNA or mRNA, directly to atarget site within an organism. For example, an agent can be locallydelivered by direct injection into a disease site such as a tumor orother target site such as a site of inflammation or a target organ suchas the liver, heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer,small intestine cancer, stomach (gastric) cancer, esophageal cancer;gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer,breast cancer, ovarian cancer; cervical cancer, prostate cancer, renalcancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein, a “tumor” comprises one or more cancerous cells.

The term “anionic precursor group” includes groups that are capable offorming an ion at physiological pH. For Example, the term includes thegroups —CO₂H, —O—P(═O)(OH)₂, —OS(═O)₂(OH), —O—S(═O)(OH), and —B(OH)₂. Inone embodiment, the anionic precursor is —CO₂H.

In a particular embodiment, PEG-C-DMA has the following structure:

wherein n is selected so that the resulting polymer chain has amolecular weight of from about 1000 to about 3000. In anotherembodiment, n is selected so that the resulting polymer chain has amolecular weight of about 2000. PEG-C-DMA can be prepared as describedby Heyes et al, Synthesis and Characterization of Novel Poly (EthyleneGlycol)-lipid Conjugates Suitable for use in Drug Delivery,” Journal ofControlled Release, 2006, and in U.S. Pat. No. 8,936,942.

DESCRIPTION OF THE EMBODIMENTS

The present invention provides novel, serum-stable lipid particlescomprising one or more active agents or therapeutic agents, methods ofmaking the lipid particles, and methods of delivering and/oradministering the lipid particles (e.g., for the treatment of a diseaseor disorder).

In one aspect, the present invention provides lipid particlescomprising: (a) one or more active agents or therapeutic agents; (b) oneor more cationic lipids comprising from about 30 mol % to about 85 mol %of the total lipid present in the particle; (c) one or more non-cationiclipids comprising from about 13 mol % to about 49.5 mol % of the totallipid present in the particle; and (d) one or more conjugated lipidsthat inhibit aggregation of particles comprising from about 0.1 mol % toabout 10 mol % of the total lipid present in the particle.

In one aspect, the present invention provides lipid particlescomprising: (a) one or more active agents or therapeutic agents; (b) oneor more cationic lipids comprising from about 50 mol % to about 85 mol %of the total lipid present in the particle; (c) one or more non-cationiclipids comprising from about 13 mol % to about 49.5 mol % of the totallipid present in the particle; and (d) one or more conjugated lipidsthat inhibit aggregation of particles comprising from about 0.5 mol % toabout 2 mol % of the total lipid present in the particle.

In certain embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particle such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In certain other embodiments, the lipid particles aresubstantially non-toxic to mammals such as humans.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, ormixtures thereof. In certain other instances, the nucleic acid comprisessingle-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid suchas, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, animmunostimulatory oligonucleotide, or mixtures thereof. In certaininstances, the nucleic acid comprises an mRNA molecule.

In other embodiments, the active agent or therapeutic agent comprises apeptide or polypeptide. In certain instances, the peptide or polypeptidecomprises an antibody such as, e.g., a polyclonal antibody, a monoclonalantibody, an antibody fragment; a humanized antibody, a recombinantantibody, a recombinant human antibody, a Primatized™ antibody, ormixtures thereof. In certain other instances, the peptide or polypeptidecomprises a cytokine, a growth factor, an apoptotic factor, adifferentiation-inducing factor, a cell-surface receptor, a ligand, ahormone, a small molecule (e.g., small organic molecule or compound), ormixtures thereof.

In one embodiment, the active agent or therapeutic agent comprises ansiRNA. In one embodiment, the siRNA molecule comprises a double-strandedregion of about 15 to about 60 nucleotides in length (e.g., about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNAmolecules of the invention are capable of silencing the expression of atarget sequence in vitro and/or in vivo.

In some embodiments, the siRNA molecule comprises at least one modifiednucleotide. In certain preferred embodiments, the siRNA moleculecomprises one, two, three, four, five, six, seven, eight, nine, ten, ormore modified nucleotides in the double-stranded region. In certaininstances, the siRNA comprises from about 1% to about 100% (e.g., about1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in thedouble-stranded region. In preferred embodiments, less than about 25%(e.g., less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% toabout 25% (e.g., from about 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%,or 10%-20%) of the nucleotides in the double-stranded region comprisemodified nucleotides.

In other embodiments, the siRNA molecule comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosinenucleotides, and mixtures thereof. In certain instances, the siRNA doesnot comprise 2′OMe-cytosine nucleotides. In other embodiments, the siRNAcomprises a hairpin loop structure.

The siRNA may comprise modified nucleotides in one strand (i.e., senseor antisense) or both strands of the double-stranded region of the siRNAmolecule. Preferably, uridine and/or guanosine nucleotides are modifiedat selective positions in the double-stranded region of the siRNAduplex. With regard to uridine nucleotide modifications, at least one,two, three, four, five, six, or more of the uridine nucleotides in thesense and/or antisense strand can be a modified uridine nucleotide suchas a 2′OMe-uridine nucleotide. In some embodiments, every uridinenucleotide in the sense and/or antisense strand is a 2′OMe-uridinenucleotide. With regard to guanosine nucleotide modifications, at leastone, two, three, four, five, six, or more of the guanosine nucleotidesin the sense and/or antisense strand can be a modified guanosinenucleotide such as a 2′OMe-guanosine nucleotide. In some embodiments,every guanosine nucleotide in the sense and/or antisense strand is a2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some preferred embodiments, a modified siRNA molecule is lessimmunostimulatory than a corresponding unmodified siRNA sequence. Insuch embodiments, the modified siRNA molecule with reducedimmunostimulatory properties advantageously retains RNAi activityagainst the target sequence. In another embodiment, theimmunostimulatory properties of the modified siRNA molecule and itsability to silence target gene expression can be balanced or optimizedby the introduction of minimal and selective 2′OMe modifications withinthe siRNA sequence such as, e.g., within the double-stranded region ofthe siRNA duplex. In certain instances, the modified siRNA is at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% less immunostimulatory than the corresponding unmodified siRNA. Itwill be readily apparent to those of skill in the art that theimmunostimulatory properties of the modified siRNA molecule and thecorresponding unmodified siRNA molecule can be determined by, forexample, measuring INF-α and/or IL-6 levels from about two to abouttwelve hours after systemic administration in a mammal or transfectionof a mammalian responder cell using an appropriate lipid-based deliverysystem (such as the LNP delivery system disclosed herein).

In certain embodiments, a modified siRNA molecule has an IC₅₀ (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC₅₀ that is less than or equal to ten-times the IC₅₀ of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC₅₀ less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC₅₀ less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC₅₀ values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of theexpression of the target sequence relative to the correspondingunmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. Preferably, the siRNAhas 3′ overhangs of two nucleotides on each side of the double-strandedregion. In certain instances, the 3′ overhang on the antisense strandhas complementarity to the target sequence and the 3′ overhang on thesense strand has complementarity to a complementary strand of the targetsequence. Alternatively, the 3′ overhangs do not have complementarity tothe target sequence or the complementary strand thereof. In someembodiments, the 3′ overhangs comprise one, two, three, four, or morenucleotides such as 2′-deoxy (2′H) nucleotides. In certain preferredembodiments, the 3′ overhangs comprise deoxythymidine (dT) and/oruridine nucleotides. In other embodiments, one or more of thenucleotides in the 3′ overhangs on one or both sides of thedouble-stranded region comprise modified nucleotides. Non-limitingexamples of modified nucleotides are described above and include 2′OMenucleotides, 2′-deoxy-2′F nucleotides, 2′-deoxy nucleotides, 2′-O-2-MOEnucleotides, LNA nucleotides, and mixtures thereof. In preferredembodiments, one, two, three, four, or more nucleotides in the 3′overhangs present on the sense and/or antisense strand of the siRNAcomprise 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosinenucleotides, and mixtures thereof.

The siRNA may comprise at least one or a cocktail (e.g., at least two,three, four, five, six, seven, eight, nine, ten, or more) of unmodifiedand/or modified siRNA sequences that silence target gene expression. Thecocktail of siRNA may comprise sequences which are directed to the sameregion or domain (e.g., a “hot spot”) and/or to different regions ordomains of one or more target genes. In certain instances, one or more(e.g., at least two, three, four, five, six, seven, eight, nine, ten, ormore) modified siRNA that silence target gene expression are present ina cocktail. In certain other instances, one or more (e.g., at least two,three, four, five, six, seven, eight, nine, ten, or more) unmodifiedsiRNA sequences that silence target gene expression are present in acocktail.

In some embodiments, the antisense strand of the siRNA moleculecomprises or consists of a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence ora portion thereof. In other embodiments, the antisense strand of thesiRNA molecule comprises or consists of a sequence that is 100%complementary to the target sequence or a portion thereof. In furtherembodiments, the antisense strand of the siRNA molecule comprises orconsists of a sequence that specifically hybridizes to the targetsequence or a portion thereof.

In further embodiments, the sense strand of the siRNA molecule comprisesor consists of a sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the target sequence or a portionthereof. In additional embodiments, the sense strand of the siRNAmolecule comprises or consists of a sequence that is 100% identical tothe target sequence or a portion thereof.

In the lipid nanoparticles of the invention, the cationic lipid can beselected from the compounds of Formula (I) as described herein.

In some embodiments, the cationic lipid may comprise from about 30 mol %to about 90 mol %, from about 30 mol % to about 85 mol %, from about 30mol % to about 80 mol %, from about 30 mol % to about 75 mol %, fromabout 30 mol % to about 70 mol %, from about 30 mol % to about 65 mol %,or from about 30 mol % to about 60 mol % of the total lipid present inthe particle.

In some embodiments, the cationic lipid may comprise from about 40 mol %to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40mol % to about 80 mol %, from about 40 mol % to about 75 mol %, fromabout 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %,or from about 40 mol % to about 60 mol % of the total lipid present inthe particle.

In other embodiments, the cationic lipid may comprise from about 55 mol% to about 90 mol %, from about 55 mol % to about 85 mol %, from about55 mol % to about 80 mol %, from about 55 mol % to about 75 mol %, fromabout 55 mol % to about 70 mol %, or from about 55 mol % to about 65 mol% of the total lipid present in the particle.

In yet other embodiments, the cationic lipid may comprise from about 60mol % to about 90 mol %, from about 60 mol % to about 85 mol %, fromabout 60 mol % to about 80 mol %, from about 60 mol % to about 75 mol %,or from about 60 mol % to about 70 mol % of the total lipid present inthe particle.

In still yet other embodiments, the cationic lipid may comprise fromabout 65 mol % to about 90 mol %, from about 65 mol % to about 85 mol %,from about 65 mol % to about 80 mol %, or from about 65 mol % to about75 mol % of the total lipid present in the particle.

In further embodiments, the cationic lipid may comprise from about 70mol % to about 90 mol %, from about 70 mol % to about 85 mol %, fromabout 70 mol % to about 80 mol %, from about 75 mol % to about 90 mol %,from about 75 mol % to about 85 mol %, or from about 80 mol % to about90 mol % of the total lipid present in the particle.

In additional embodiments, the cationic lipid may comprise (at least)about 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mol % (or any fractionthereof or range therein) of the total lipid present in the particle.

In the lipid particles of the invention, the non-cationic lipid maycomprise, e.g., one or more anionic lipids and/or neutral lipids. Inpreferred embodiments, the non-cationic lipid comprises one of thefollowing neutral lipid components: (1) cholesterol or a derivativethereof (2) a phospholipid; or (3) a mixture of a phospholipid andcholesterol or a derivative thereof.

Examples of cholesterol derivatives include, but are not limited to,cholestanol, cholestanone, cholestenone, coprostanol,cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether,and mixtures thereof. The synthesis of cholesteryl-2′-hydroxyethyl etheris described herein.

The phospholipid may be a neutral lipid including, but not limited to,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine(EPC), and mixtures thereof. In certain preferred embodiments, thephospholipid is DPPC, DSPC, or mixtures thereof.

In some embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol% to about 60 mol %, from about 25 mol % to about 60 mol %, from about30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, fromabout 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %,from about 25 mol % to about 55 mol %, from about 30 mol % to about 55mol %, from about 13 mol % to about 50 mol %, from about 15 mol % toabout 50 mol % or from about 20 mol % to about 50 mol % of the totallipid present in the particle. When the non-cationic lipid is a mixtureof a phospholipid and cholesterol or a cholesterol derivative, themixture may comprise up to about 40, 50, or 60 mol % of the total lipidpresent in the particle.

In other embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 49.5 mol %, from about 13 mol % to about 49.5 mol %, from about 15mol % to about 49.5 mol %, from about 20 mol % to about 49.5 mol %, fromabout 25 mol % to about 49.5 mol %, from about 30 mol % to about 49.5mol %, from about 35 mol % to about 49.5 mol %, or from about 40 mol %to about 49.5 mol % of the total lipid present in the particle.

In yet other embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 45 mol %, from about 13 mol % to about 45 mol %, from about 15 mol% to about 45 mol %, from about 20 mol % to about 45 mol %, from about25 mol % to about 45 mol %, from about 30 mol % to about 45 mol %, orfrom about 35 mol % to about 45 mol % of the total lipid present in theparticle.

In still yet other embodiments, the non-cationic lipid (e.g., one ormore phospholipids and/or cholesterol) may comprise from about 10 mol %to about 40 mol %, from about 13 mol % to about 40 mol %, from about 15mol % to about 40 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 40 mol %, or from about 30 mol % to about 40 mol% of the total lipid present in the particle.

In further embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 35 mol %, from about 13 mol % to about 35 mol %, from about 15 mol% to about 35 mol %, from about 20 mol % to about 35 mol 25%, or fromabout 25 mol % to about 35 mol % of the total lipid present in theparticle.

In yet further embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 30 mol %, from about 13 mol % to about 30 mol %, from about 15 mol% to about 30 mol %, from about 20 mol % to about 30 mol %, from about10 mol % to about 25 mol %, from about 13 mol % to about 25 mol %, orfrom about 15 mol % to about 25 mol % of the total lipid present in theparticle.

In additional embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise (at least) about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle.

In certain preferred embodiments, the non-cationic lipid comprisescholesterol or a derivative thereof of from about 31.5 mol % to about42.5 mol % of the total lipid present in the particle. As a non-limitingexample, a phospholipid-free lipid particle of the invention maycomprise cholesterol or a derivative thereof at about 37 mol % of thetotal lipid present in the particle. In other preferred embodiments, aphospholipid-free lipid particle of the invention may comprisecholesterol or a derivative thereof of from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 30 mol % toabout 35 mol %, from about 35 mol % to about 45 mol %, from about 40 mol% to about 45 mol %, from about 32 mol % to about 45 mol %, from about32 mol % to about 42 mol %, from about 32 mol % to about 40 mol %, fromabout 34 mol % to about 45 mol %, from about 34 mol % to about 42 mol %,from about 34 mol % to about 40 mol %, or about 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereofor range therein) of the total lipid present in the particle.

In certain other preferred embodiments, the non-cationic lipid comprisesa mixture of: (i) a phospholipid of from about 4 mol % to about 10 mol %of the total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 30 mol % to about 40 mol % of the totallipid present in the particle. As a non-limiting example, a lipidparticle comprising a mixture of a phospholipid and cholesterol maycomprise DPPC at about 7 mol % and cholesterol at about 34 mol % of thetotal lipid present in the particle. In other embodiments, thenon-cationic lipid comprises a mixture of: (i) a phospholipid of fromabout 3 mol % to about 15 mol %, from about 4 mol % to about 15 mol %,from about 4 mol % to about 12 mol %, from about 4 mol % to about 10 mol%, from about 4 mol % to about 8 mol %, from about 5 mol % to about 12mol %, from about 5 mol % to about 9 mol %, from about 6 mol % to about12 mol %, from about 6 mol % to about 10 mol %, or about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle; and (ii)cholesterol or a derivative thereof of from about 25 mol % to about 45mol %, from about 30 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 40 mol %, from about 25 mol% to about 35 mol %, from about 30 mol % to about 35 mol %, from about35 mol % to about 45 mol %, from about 40 mol % to about 45 mol %, fromabout 28 mol % to about 40 mol %, from about 28 mol % to about 38 mol %,from about 30 mol % to about 38 mol %, from about 32 mol % to about 36mol %, or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In further preferred embodiments, the non-cationic lipid comprises amixture of: (i) a phospholipid of from about 10 mol % to about 30 mol %of the total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 10 mol % to about 30 mol % of the totallipid present in the particle. As a non-limiting example, a lipidparticle comprising a mixture of a phospholipid and cholesterol maycomprise DPPC at about 20 mol % and cholesterol at about 20 mol % of thetotal lipid present in the particle. In other embodiments, thenon-cationic lipid comprises a mixture of: (i) a phospholipid of fromabout 10 mol % to about 30 mol %, from about 10 mol % to about 25 mol %,from about 10 mol % to about 20 mol %, from about 15 mol % to about 30mol %, from about 20 mol % to about 30 mol %, from about 15 mol % toabout 25 mol %, from about 12 mol % to about 28 mol %, from about 14 mol% to about 26 mol %, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol % (or any fractionthereof or range therein) of the total lipid present in the particle;and (ii) cholesterol or a derivative thereof of from about 10 mol % toabout 30 mol %, from about 10 mol % to about 25 mol %, from about 10 mol% to about 20 mol %, from about 15 mol % to about 30 mol %, from about20 mol % to about 30 mol %, from about 15 mol % to about 25 mol %, fromabout 12 mol % to about 28 mol %, from about 14 mol % to about 26 mol %,or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 mol % (or any fraction thereof or range therein)of the total lipid present in the particle.

Conjugated Lipid

In the lipid particles of the invention (e.g., LNP comprising, e.g., aninterfering RNA such as siRNA, or mRNA), the conjugated lipid maycomprise, e.g., one or more of the following: a polyethyleneglycol(PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, or mixturesthereof. In one preferred embodiment, the nucleic acid-lipid particlescomprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. Theconjugated lipid s may comprise a PEG-lipid including, e.g., aPEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAAconjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl(C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18),or mixtures thereof.

Additional PEG-lipid conjugates suitable for use in the inventioninclude, but are not limited to,mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). Thesynthesis of PEG-C-DOMG is described in PCT Application No.PCT/US08/88676, filed Dec. 31, 2008, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Yetadditional PEG-lipid conjugates suitable for use in the inventioninclude, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-w-methyl-poly(ethyleneglycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the conjugated lipid (e.g., PEG-lipid conjugate)may comprise from about 0.1 to about 10% (or any fraction thereof orrange therein) of the total lipid present in the particle. In certaininstances, the conjugated lipid (e.g., PEG-lipid conjugate) may comprisefrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, fromabout 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % toabout 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

In the lipid particles of the invention, the active agent or therapeuticagent may be fully encapsulated within the lipid portion of theparticle, thereby protecting the active agent or therapeutic agent fromenzymatic degradation. In preferred embodiments, a LNP comprising anucleic acid, such as an interfering RNA (e.g., siRNA) or mRNA, is fullyencapsulated within the lipid portion of the particle, therebyprotecting the nucleic acid from nuclease degradation. In certaininstances, the nucleic acid in the LNP is not substantially degradedafter exposure of the particle to a nuclease at 37° C. for at leastabout 20, 30, 45, or 60 minutes. In certain other instances, the nucleicacid in the LNP is not substantially degraded after incubation of theparticle in serum at 37° C. for at least about 30, 45, or 60 minutes orat least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agentor therapeutic agent (e.g., nucleic acid such as siRNA) is complexedwith the lipid portion of the particle. One of the benefits of theformulations of the present invention is that the lipid particlecompositions are substantially non-toxic to mammals such as humans.

The term “fully encapsulated” indicates that the active agent ortherapeutic agent in the lipid particle is not significantly degradedafter exposure to serum or a nuclease or protease assay that wouldsignificantly degrade free DNA, RNA, or protein. In a fully encapsulatedsystem, preferably less than about 25% of the active agent ortherapeutic agent in the particle is degraded in a treatment that wouldnormally degrade 100% of free active agent or therapeutic agent, morepreferably less than about 10%, and most preferably less than about 5%of the active agent or therapeutic agent in the particle is degraded. Inthe context of nucleic acid therapeutic agents, full encapsulation maybe determined by an Oligreen® assay. Oligreen® is an ultra-sensitivefluorescent nucleic acid stain for quantitating oligonucleotides andsingle-stranded DNA or RNA in solution (available from InvitrogenCorporation; Carlsbad, Calif.). “Fully encapsulated” also indicates thatthe lipid particles are serum-stable, that is, that they do not rapidlydecompose into their component parts upon in vivo administration.

In another aspect, the present invention provides a lipid particle(e.g., LNP) composition comprising a plurality of lipid particles. Inpreferred embodiments, the active agent or therapeutic agent (e.g.,nucleic acid) is fully encapsulated within the lipid portion of thelipid particles (e.g., LNP), such that from about 30% to about 100%,from about 40% to about 100%, from about 50% to about 100%, from about60% to about 100%, from about 70% to about 100%, from about 80% to about100%, from about 90% to about 100%, from about 30% to about 95%, fromabout 40% to about 95%, from about 50% to about 95%, from about 60% toabout 95%, %, from about 70% to about 95%, from about 80% to about 95%,from about 85% to about 95%, from about 90% to about 95%, from about 30%to about 90%, from about 40% to about 90%, from about 50% to about 90%,from about 60% to about 90%, from about 70% to about 90%, from about 80%to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%(or any fraction thereof or range therein) of the lipid particles (e.g.,LNP) have the active agent or therapeutic agent encapsulated therein.

Typically, the lipid particles (e.g., LNP) of the invention have alipid:active agent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) offrom about 1 to about 100. In some instances, the lipid:active agent(e.g., lipid:nucleic acid) ratio (mass/mass ratio) ranges from about 1to about 50, from about 2 to about 25, from about 3 to about 20, fromabout 4 to about 15, or from about 5 to about 10. In preferredembodiments, the lipid particles of the invention have a lipid:activeagent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) of from about 5to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (orany fraction thereof or range therein).

Typically, the lipid particles (e.g., LNP) of the invention have a meandiameter of from about 40 nm to about 150 nm. In preferred embodiments,the lipid particles (e.g., LNP) of the invention have a mean diameter offrom about 40 nm to about 130 nm, from about 40 nm to about 120 nm, fromabout 40 nm to about 100 nm, from about 50 nm to about 120 nm, fromabout 50 nm to about 100 nm, from about 60 nm to about 120 nm, fromabout 60 nm to about 110 nm, from about 60 nm to about 100 nm, fromabout 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about70 nm to about 120 nm, from about 70 nm to about 110 nm, from about 70nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm toabout 80 nm, or less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm(or any fraction thereof or range therein).

In one specific embodiment of the invention, the LNP comprises: (a) oneor more unmodified and/or modified nucleic acid molecules (e.g.,interfering RNA that silence target gene expression, such as siRNA,aiRNA, miRNA; or mRNA that result in target protein expression); (b) acationic lipid comprising from about 56.5 mol % to about 66.5 mol % ofthe total lipid present in the particle; (c) a non-cationic lipidcomprising from about 31.5 mol % to about 42.5 mol % of the total lipidpresent in the particle; and (d) a conjugated lipid that inhibitsaggregation of particles comprising from about 1 mol % to about 2 mol %of the total lipid present in the particle. This specific embodiment ofLNP is generally referred to herein as the “1:62” formulation. In apreferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA(“XTC2”), the non-cationic lipid is cholesterol, and the conjugatedlipid is a PEG-DAA conjugate. Although these are preferred embodimentsof the 1:62 formulation, those of skill in the art will appreciate thatother cationic lipids, non-cationic lipids (including other cholesterolderivatives), and conjugated lipids can be used in the 1:62 formulationas described herein.

In another specific embodiment of the invention, the LNP comprises: (a)one or more unmodified and/or modified nucleic acid molecules (e.g.,interfering RNA that silence target gene expression, such as siRNA,aiRNA, miRNA; or mRNA that result in target protein expression); (b) acationic lipid comprising from about 52 mol % to about 62 mol % of thetotal lipid present in the particle; (c) a non-cationic lipid comprisingfrom about 36 mol % to about 47 mol % of the total lipid present in theparticle; and (d) a conjugated lipid that inhibits aggregation ofparticles comprising from about 1 mol % to about 2 mol % of the totallipid present in the particle. This specific embodiment of LNP isgenerally referred to herein as the “1:57” formulation. In one preferredembodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), thenon-cationic lipid is a mixture of a phospholipid (such as DPPC) andcholesterol, wherein the phospholipid comprises from about 5 mol % toabout 9 mol % of the total lipid present in the particle (e.g., about7.1 mol %) and the cholesterol (or cholesterol derivative) comprisesfrom about 32 mol % to about 37 mol % of the total lipid present in theparticle (e.g., about 34.3 mol %), and the PEG-lipid is a PEG-DAA (e.g.,PEG-cDMA). In another preferred embodiment, the cationic lipid isDLinDMA or DLin-K-C2-DMA (“XTC2”), the non-cationic lipid is a mixtureof a phospholipid (such as DPPC) and cholesterol, wherein thephospholipid comprises from about 15 mol % to about 25 mol % of thetotal lipid present in the particle (e.g., about 20 mol %) and thecholesterol (or cholesterol derivative) comprises from about 15 mol % toabout 25 mol % of the total lipid present in the particle (e.g., about20 mol %), and the PEG-lipid is a PEG-DAA (e.g., PEG-cDMA). Althoughthese are preferred embodiments of the 1:57 formulation, those of skillin the art will appreciate that other cationic lipids, non-cationiclipids (including other phospholipids and other cholesterolderivatives), and conjugated lipids can be used in the 1:57 formulationas described herein.

In preferred embodiments, the 1:62 LNP formulation is a three-componentsystem which is phospholipid-free and comprises about 1.5 mol % PEG-cDMA(or PEG-IDSA), about 61.5 mol % DLinDMA (or XTC2), and about 36.9 mol %cholesterol (or derivative thereof). In other preferred embodiments, the1:57 LNP formulation is a four-component system which comprises about1.4 mol % PEG-cDMA (or PEG-cDSA), about 57.1 mol % DLinDMA (or XTC2),about 7.1 mol % DPPC, and about 34.3 mol % cholesterol (or derivativethereof). In yet other preferred embodiments, the 1:57 LNP formulationis a four-component system which comprises about 1.4 mol % PEG-cDMA (orPEG-cDSA), about 57.1 mol % DLinDMA (or XTC2), about 20 mol % DPPC, andabout 20 mol % cholesterol (or derivative thereof). It should beunderstood that these LNP formulations are target formulations, and thatthe amount of lipid (both cationic and non-cationic) present and theamount of lipid conjugate present in the LNP formulations may vary.

The present invention also provides a pharmaceutical compositioncomprising a lipid particle (e.g., LNP) described herein and apharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method forintroducing one or more active agents or therapeutic agents (e.g.,nucleic acid) into a cell, comprising contacting the cell with a lipidparticle (e.g., LNP) described herein. In one embodiment, the cell is ina mammal and the mammal is a human. In another embodiment, the presentinvention provides a method for the in vivo delivery of one or moreactive agents or therapeutic agents (e.g., nucleic acid), comprisingadministering to a mammalian subject a lipid particle (e.g., LNP)described herein. In a preferred embodiment, the mode of administrationincludes, but is not limited to, oral, intranasal, intravenous,intraperitoneal, intramuscular, intra-articular, intralesional,intratracheal, subcutaneous, and intradermal. Preferably, the mammaliansubject is a human.

In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the totalinjected dose of the lipid particles (e.g., LNP) is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles (e.g., LNP) is present inplasma about 8, 12, 24, 36, or 48 hours after injection. In certaininstances, more than about 10% of a plurality of the particles ispresent in the plasma of a mammal about 1 hour after administration. Incertain other instances, the presence of the lipid particles (e.g., LNP)is detectable at least about 1 hour after administration of theparticle. In certain embodiments, the presence of an active agent ortherapeutic agent, such as an interfering RNA (e.g., siRNA) or mRNA isdetectable in cells of the at about 8, 12, 24, 36, 48, 60, 72 or 96hours after administration (e.g., lung, liver, tumor, or at a site ofinflammation). In other embodiments, downregulation of expression of atarget sequence by an active agent or therapeutic agent, such as aninterfering RNA (e.g., siRNA) is detectable at about 8, 12, 24, 36, 48,60, 72 or 96 hours after administration. In yet other embodiments,downregulation of expression of a target sequence by an active agent ortherapeutic agent such as an interfering RNA (e.g., siRNA) occurspreferentially in tumor cells or in cells at a site of inflammation. Infurther embodiments, the presence or effect of an active agent ortherapeutic agent such as an interfering RNA (e.g., siRNA) in cells at asite proximal or distal to the site of administration or in cells of thelung, liver, or a tumor is detectable at about 12, 24, 48, 72, or 96hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28days after administration. In other embodiments, upregulation ofexpression of a target sequence by an active agent or therapeutic agent,such as an mRNA or self-amplifying RNA is detectable at about 8, 12, 24,36, 48, 60, 72 or 96 hours after administration. In yet otherembodiments, upregulation of expression of a target sequence by anactive agent or therapeutic agent such as an mRNA or self-amplifying RNAoccurs preferentially in tumor cells or in cells at a site ofinflammation. In further embodiments, the presence or effect of anactive agent or therapeutic agent such as an mRNA or self-replicatingRNA in cells at a site proximal or distal to the site of administrationor in cells of the lung, liver, or a tumor is detectable at about 12,24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20,22, 24, 26, or 28 days after administration. In additional embodiments,the lipid particles (e.g., LNP) of the invention are administeredparenterally or intraperitoneally.

In some embodiments, the lipid particles (e.g., LNP) of the inventionare particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle (e.g., LNP) maybe administered to the mammal. In some instances, an interfering RNA(e.g., siRNA) is formulated into a LNP, and the particles areadministered to patients requiring such treatment. In other instances,cells are removed from a patient, the interfering RNA (e.g., siRNA) isdelivered in vitro (e.g., using a LNP described herein), and the cellsare reinjected into the patient.

In an additional aspect, the present invention provides lipid particles(e.g., LNP) comprising asymmetrical interfering RNA (aiRNA) moleculesthat silence the expression of a target gene and methods of using suchparticles to silence target gene expression.

In one embodiment, the aiRNA molecule comprises a double-stranded(duplex) region of about 10 to about 25 (base paired) nucleotides inlength, wherein the aiRNA molecule comprises an antisense strandcomprising 5′ and 3′ overhangs, and wherein the aiRNA molecule iscapable of silencing target gene expression.

In certain instances, the aiRNA molecule comprises a double-stranded(duplex) region of about 12-20, 12-19, 12-18, 13-17, or 14-17 (basepaired) nucleotides in length, more typically 12, 13, 14, 15, 16, 17,18, 19, or 20 (base paired) nucleotides in length. In certain otherinstances, the 5′ and 3′ overhangs on the antisense strand comprisesequences that are complementary to the target RNA sequence, and mayoptionally further comprise nontargeting sequences. In some embodiments,each of the 5′ and 3′ overhangs on the antisense strand comprises orconsists of one, two, three, four, five, six, seven, or morenucleotides.

In other embodiments, the aiRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In a preferred embodiment, the aiRNAmolecule comprises 2′OMe nucleotides. As a non-limiting example, the2′OMe nucleotides may be selected from the group consisting of2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, and mixturesthereof.

In a related aspect, the present invention provides lipid particles(e.g., LNP) comprising microRNA (miRNA) molecules that silence theexpression of a target gene and methods of using such compositions tosilence target gene expression.

In one embodiment, the miRNA molecule comprises about 15 to about 60nucleotides in length, wherein the miRNA molecule is capable ofsilencing target gene expression.

In certain instances, the miRNA molecule comprises about 15-50, 15-40,or 15-30 nucleotides in length, more typically about 15-25 or 19-25nucleotides in length, and are preferably about 20-24, 21-22, or 21-23nucleotides in length. In a preferred embodiment, the miRNA molecule isa mature miRNA molecule targeting an RNA sequence of interest.

In some embodiments, the miRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In a preferred embodiment, the miRNAmolecule comprises 2′OMe nucleotides. As a non-limiting example, the2′OMe nucleotides may be selected from the group consisting of2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, and mixturesthereof.

In some embodiments, the lipid particles (e.g., LNP) of the inventionare useful in methods for the therapeutic delivery of one or more mRNAmolecules. In particular, it is one object of this invention to providein vitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) through the expression of one or more targetproteins. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of one or more mRNA molecules to a mammaliansubject. In certain other embodiments, a therapeutically effectiveamount of the lipid particle (e.g., LNP) may be administered to themammal. In some instances, one or more mRNA molecules are formulatedinto a LNP, and the particles are administered to patients requiringsuch treatment. In other instances, cells are removed from a patient,one or more mRNA molecules are delivered in vitro (e.g., using a LNPdescribed herein), and the cells are reinjected into the patient.

In other embodiments, the mRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In a related aspect, the presentinvention provides lipid particles (e.g., LNP) comprising microRNA(miRNA) molecules that silence the expression of a target gene andmethods of using such compositions to silence target gene expression.

As such, the lipid particles of the invention (e.g., LNP) areadvantageous and suitable for use in the administration of active agentsor therapeutic agents, such as nucleic acid (e.g., interfering RNA suchas siRNA, aiRNA, and/or miRNA; or mRNA) to a subject (e.g., a mammalsuch as a human) because they are stable in circulation, of a sizerequired for pharmacodynamic behavior resulting in access toextravascular sites, and are capable of reaching target cellpopulations.

Active Agents

Active agents (e.g., therapeutic agents) include any molecule orcompound capable of exerting a desired effect on a cell, tissue, organ,or subject. Such effects may be, e.g., biological, physiological, and/orcosmetic. Active agents may be any type of molecule or compoundincluding, but not limited to, nucleic acids, peptides, polypeptides,small molecules, and mixtures thereof. Non-limiting examples of nucleicacids include interfering RNA molecules (e.g., siRNA, aiRNA, miRNA),antisense oligonucleotides, mRNA, self-amplifying RNA, plasmids,ribozymes, immunostimulatory oligonucleotides, and mixtures thereof.Examples of peptides or polypeptides include, without limitation,antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, Primatized™ antibodies), cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell-surfacereceptors and their ligands, hormones, and mixtures thereof. Examples ofsmall molecules include, but are not limited to, small organic moleculesor compounds such as any conventional agent or drug known to those ofskill in the art.

In some embodiments, the active agent is a therapeutic agent, or a saltor derivative thereof. Therapeutic agent derivatives may betherapeutically active themselves or they may be prodrugs, which becomeactive upon further modification. Thus, in one embodiment, a therapeuticagent derivative retains some or all of the therapeutic activity ascompared to the unmodified agent, while in another embodiment, atherapeutic agent derivative is a prodrug that lacks therapeuticactivity, but becomes active upon further modification.

Nucleic Acids

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle (e.g., LNP). In some embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” includes any oligonucleotide or polynucleotide, with fragmentscontaining up to 60 nucleotides generally termed oligonucleotides, andlonger fragments termed polynucleotides. In particular embodiments,oligonucleotides of the invention are from about 15 to about 60nucleotides in length. Nucleic acid may be administered alone in thelipid particles of the invention, or in combination (e.g.,co-administered) with lipid particles of the invention comprisingpeptides, polypeptides, or small molecules such as conventional drugs.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA are described herein and include, e.g., structuralgenes, genes including control and termination regions, andself-replicating systems such as viral or plasmid DNA. Examples ofdouble-stranded RNA are described herein and include, e.g., siRNA andother RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleicacids include, e.g., antisense oligonucleotides, ribozymes, maturemiRNA, and triplex-forming oligonucleotides.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In preferred embodiments,an oligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressiontherefrom, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

siRNA

The siRNA component of the nucleic acid-lipid particles of the presentinvention is capable of silencing the expression of a target gene ofinterest. Each strand of the siRNA duplex is typically about 15 to about60 nucleotides in length, preferably about 15 to about 30 nucleotides inlength. In certain embodiments, the siRNA comprises at least onemodified nucleotide. The modified siRNA is generally lessimmunostimulatory than a corresponding unmodified siRNA sequence andretains RNAi activity against the target gene of interest. In someembodiments, the modified siRNA contains at least one 2′OMe purine orpyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine,2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. In preferredembodiments, one or more of the uridine and/or guanosine nucleotides aremodified. The modified nucleotides can be present in one strand (i.e.,sense or antisense) or both strands of the siRNA. The siRNA sequencesmay have overhangs (e.g., 3′ or 5′ overhangs as described in Elbashir etal., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)),or may lack overhangs (i.e., have blunt ends).

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) modified nucleotides in the double-stranded region ofthe siRNA duplex. In certain embodiments, one, two, three, four, five,six, seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides.

In some embodiments, less than about 25% (e.g., less than about 25%,24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides.

In other embodiments, from about 1% to about 25% (e.g., from about1%-25%, 2%-25%, 3%-25%, 4%-25%, 5%-25%, 6%-25%, 7%-25%, 8%-25%, 9%-25%,10%-25%, 11%-25%, 12%-25%, 13%-25%, 14%-25%, 15%-25%, 16%-25%, 17%-25%,18%-25%, 19%-25%, 20%-25%, 21%-25%, 22%-25%, 23%-25%, 24%-25%, etc.) orfrom about 1% to about 20% (e.g., from about 1%-20%, 2%-20%, 3%-20%,4%-20%, 5%-20%, 6%-20%, 7%-20%, 8%-20%, 9%-20%, 10%-20%, 11%-20%,12%-20%, 13%-20%, 14%-20%, 15%-20%, 16%-20%, 17%-20%, 18%-20%, 19%-20%,1%-19%, 2%-19%, 3%-19%, 4%-19%, 5%-19%, 6%-19%, 7%-19%, 8%-19%, 9%-19%,10%-19%, 11%-19%, 12%-19%, 13%-19%, 14%-19%, 15%-19%, 16%-19%, 17%-19%,18%-19%, 1%-18%, 2%-18%, 3%-18%, 4%-18%, 5%-18%, 6%-18%, 7%-18%, 8%-18%,9%-18%, 10%-18%, 11%-18%, 12%-18%, 13%-18%, 14%-18%, 15%-18%, 16%-18%,17%-18%, 1%-17%, 2%-17%, 3%-17%, 4%-17%, 5%-17%, 6%-17%, 7%-17%, 8%-17%,9%-17%, 10%-17%, 11%-17%, 12%-17%, 13%-17%, 14%-17%, 15%-17%, 16%-17%,1%-16%, 2%-16%, 3%-16%, 4%-16%, 5%-16%, 6%-16%, 7%-16%, 8%-16%, 9%-16%,10%-16%, 11%-16%, 12%-16%, 13%-16%, 14%-16%, 15%-16%, 1%-15%, 2%-15%,3%-15%, 4%-15%, 5%-15%, 6%-15%, 7%-15%, 8%-15%, 9%-15%, 10%-15%,11%-15%, 12%-15%, 13%-15%, 14%-15%, etc.) of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides.

In further embodiments, e.g., when one or both strands of the siRNA areselectively modified at uridine and/or guanosine nucleotides, theresulting modified siRNA can comprise less than about 30% modifiednucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%,23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% modified nucleotides) or fromabout 1% to about 30% modified nucleotides (e.g., from about 1%-30%,2%-30%, 3%-30%, 4%-30%, 5%-30%, 6%-30%, 7%-30%, 8%-30%, 9%-30%, 10%-30%,11%-30%, 12%-30%, 13%-30%, 14%-30%, 15%-30%, 16%-30%, 17%-30%, 18%-30%,19%-30%, 20%-30%, 21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 26%-30%,27%-30%, 28%-30%, or 29%-30% modified nucleotides).

Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22(3):326-330 (2004).

Generally, the nucleotide sequence 3′ of the AUG start codon of atranscript from the target gene of interest is scanned for dinucleotidesequences (e.g., AA, NA, CC, GG, or UU, wherein N=C, G, or U) (see,e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotidesimmediately 3′ to the dinucleotide sequences are identified as potentialsiRNA sequences (i.e., a target sequence or a sense strand sequence).Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotidesimmediately 3′ to the dinucleotide sequences are identified as potentialsiRNA sequences. In some embodiments, the dinucleotide sequence is an AAor NA sequence and the 19 nucleotides immediately 3′ to the AA or NAdinucleotide are identified as potential siRNA sequences. siRNAsequences are usually spaced at different positions along the length ofthe target gene. To further enhance silencing efficiency of the siRNAsequences, potential siRNA sequences may be analyzed to identify sitesthat do not contain regions of homology to other coding sequences, e.g.,in the target cell or organism. For example, a suitable siRNA sequenceof about 21 base pairs typically will not have more than 16-17contiguous base pairs of homology to coding sequences in the target cellor organism. If the siRNA sequences are to be expressed from an RNA PolIII promoter, siRNA sequences lacking more than 4 contiguous A's or T'sare selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g., http://boz094.ust.hk/RNAi/siRNA. One ofskill in the art will appreciate that sequences with one or more of theforegoing characteristics may be selected for further analysis andtesting as potential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available athttp://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′,5′-UGU-3′,5′-GUGU-3′,5′-UGUGU-3′, etc.) can alsoprovide an indication of whether the sequence may be immunostimulatory.Once an siRNA molecule is found to be immunostimulatory, it can then bemodified to decrease its immunostimulatory properties as describedherein. As a non-limiting example, an siRNA sequence can be contactedwith a mammalian responder cell under conditions such that the cellproduces a detectable immune response to determine whether the siRNA isan immunostimulatory or a non-immunostimulatory siRNA. The mammalianresponder cell may be from a naïve mammal (i.e., a mammal that has notpreviously been in contact with the gene product of the siRNA sequence).The mammalian responder cell may be, e.g., a peripheral bloodmononuclear cell (PBMC), a macrophage, and the like. The detectableimmune response may comprise production of a cytokine or growth factorsuch as, e.g., TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or a combinationthereof. An siRNA molecule identified as being immunostimulatory canthen be modified to decrease its immunostimulatory properties byreplacing at least one of the nucleotides on the sense and/or antisensestrand with modified nucleotides. For example, less than about 30%(e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of thenucleotides in the double-stranded region of the siRNA duplex can bereplaced with modified nucleotides such as 2′OMe nucleotides. Themodified siRNA can then be contacted with a mammalian responder cell asdescribed above to confirm that its immunostimulatory properties havebeen reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α (PBLBiomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; SanDiego, Calif.); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; SanDiego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. The siRNA sequences may haveoverhangs (e.g., 3′ or 5′ overhangs as described in Elbashir et al.,Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001), ormay lack overhangs (i.e., to have blunt ends).

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Preferably, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses atthe 0.2 μmol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis platforms as wellas large scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, siRNA molecules can be assembledfrom two distinct oligonucleotides, wherein one oligonucleotidecomprises the sense strand and the other comprises the antisense strandof the siRNA. For example, each strand can be synthesized separately andjoined together by hybridization or ligation following synthesis and/ordeprotection. In certain other instances, siRNA molecules can besynthesized as a single continuous oligonucleotide fragment, where theself-complementary sense and antisense regions hybridize to form ansiRNA duplex having hairpin secondary structure.

Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In preferred embodiments,the degree of chemical modifications introduced into the siRNA moleculestrikes a balance between reduction or abrogation of theimmunostimulatory properties of the siRNA and retention of RNAiactivity. As a non-limiting example, an siRNA molecule that targets agene of interest can be minimally modified (e.g., less than about 30%,25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/orguanosine nucleotides within the siRNA duplex to eliminate the immuneresponse generated by the siRNA while retaining its capability tosilence target gene expression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl) nucleotides, and 2′-azidonucleotides. In certain instances, the siRNA molecules described hereininclude one or more G-clamp nucleotides. A G-clamp nucleotide refers toa modified cytosine analog wherein the modifications confer the abilityto hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (see, e.g., Lin et al.,J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotideshaving a nucleotide base analog such as, for example, C-phenyl,C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res.,29:2437-2447 (2001)) can be incorporated into siRNA molecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides and/or anycombination of modified and unmodified nucleotides. Additional examplesof modified nucleotides and types of chemical modifications that can beintroduced into siRNA molecules are described, e.g., in UK Patent No. GB2,397,818 B and U.S. Patent Publication Nos. 20040192626, 20050282188,and 20070135372, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the l′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). In certaininstances, the conjugate is a molecule that facilitates the delivery ofthe siRNA into a cell. Examples of conjugate molecules suitable forattachment to siRNA include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Yet other examples includethe 2′-O-alkyl amine, 2′-ρ-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples include the hydrophobicgroup, membrane active compound, cell penetrating compound, celltargeting signal, interaction modifier, and steric stabilizer conjugatemolecules described in U.S. Patent Publication No. 20040167090. Furtherexamples include the conjugate molecules described in U.S. PatentPublication No. 20050239739. The type of conjugate used and the extentof conjugation to the siRNA molecule can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of the siRNAwhile retaining RNAi activity. As such, one skilled in the art canscreen siRNA molecules having various conjugates attached thereto toidentify ones having improved properties and full RNAi activity usingany of a variety of well-known in vitro cell culture or in vivo animalmodels. The disclosures of the above-described patent documents areherein incorporated by reference in their entirety for all purposes.

Target Genes

In certain embodiments, the nucleic acid component (e.g., siRNA) of thenucleic acid-lipid particles described herein can be used todownregulate or silence the translation (i.e., expression) of a gene ofinterest. Genes of interest include, but are not limited to, genesassociated with viral infection and survival, genes associated withmetabolic diseases and disorders (e.g., liver diseases and disorders),genes associated with tumorigenesis and cell transformation (e.g.,cancer), angiogenic genes, immunomodulator genes such as thoseassociated with inflammatory and autoimmune responses, ligand receptorgenes, and genes associated with neurodegenerative disorders. In certainembodiments, the gene of interest is expressed in hepatocytes.

Genes associated with viral infection and survival include thoseexpressed by a virus in order to bind, enter, and replicate in a cell.Of particular interest are viral sequences associated with chronic viraldiseases. Viral sequences of particular interest include sequences ofFiloviruses such as Ebola virus and Marburg virus (see, e.g., Geisbertet al., J. Infect. Dis., 193:1650-1657 (2006)); Arenaviruses such asLassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabiavirus (Buchmeier et al., Arenaviridae: the viruses and theirreplication, In: FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed.,Lippincott-Raven, Philadelphia, (2001)); Influenza viruses such asInfluenza A, B, and C viruses, (see, e.g., Steinhauer et al., Annu RevGenet., 36:305-332 (2002); and Neumann et al., J Gen Virol.,83:2635-2662 (2002)); Hepatitis viruses (see, e.g., Hamasaki et al.,FEBS Lett., 543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003);Schlomai et al., Hepatology, 37:764 (2003); Wilson et al., Proc. Natl.Acad. Sci. USA, 100:2783 (2003); Kapadia et al., Proc. Natl. Acad. Sci.USA, 100:2014 (2003); and FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed.,Lippincott-Raven, Philadelphia (2001)); Human Immunodeficiency Virus(HIV) (Banerjea et al., Mol. Ther., 8:62 (2003); Song et al., J. Virol.,77:7174 (2003); Stephenson, JAMA, 289:1494 (2003); Qin et al., Proc.Natl. Acad. Sci. USA, 100:183 (2003)); Herpes viruses (Jia et al., J.Virol., 77:3301 (2003)); and Human Papilloma Viruses (HPV) (Hall et al.,J. Virol., 77:6066 (2003); Jiang et al., Oncogene, 21:6041 (2002)).

Exemplary Filovirus nucleic acid sequences that can be silenced include,but are not limited to, nucleic acid sequences encoding structuralproteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein(L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein(GP), VP24). Complete genome sequences for Ebola virus are set forth in,e.g., Genbank Accession Nos. NC_002549; AY769362; NC_006432; NC_004161;AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001;and AF086833. Ebola virus VP24 sequences are set forth in, e.g., GenbankAccession Nos. U77385 and AY058897. Ebola virus L-pol sequences are setforth in, e.g., Genbank Accession No. X67110. Ebola virus VP40 sequencesare set forth in, e.g., Genbank Accession No. AY058896. Ebola virus NPsequences are set forth in, e.g., Genbank Accession No. AY058895. Ebolavirus GP sequences are set forth in, e.g., Genbank Accession No.AY058898; Sanchez et al., Virus Res., 29:215-240 (1993); Will et al., J.Virol., 67:1203-1210 (1993); Volchkov et al., FEBS Lett., 305:181-184(1992); and U.S. Pat. No. 6,713,069. Additional Ebola virus sequencesare set forth in, e.g., Genbank Accession Nos. L11365 and X61274.Complete genome sequences for Marburg virus are set forth in, e.g.,Genbank Accession Nos. NC_001608; AY430365; AY430366; and AY358025.Marburg virus GP sequences are set forth in, e.g., Genbank AccessionNos. AF005734; AF005733; and AF005732. Marburg virus VP35 sequences areset forth in, e.g., Genbank Accession Nos. AF005731 and AF005730.Additional Marburg virus sequences are set forth in, e.g., GenbankAccession Nos. X64406; Z29337; AF005735; and Z12132. Non-limitingexamples of siRNA molecules targeting Ebola virus and Marburg virusnucleic acid sequences include those described in U.S. PatentPublication No. 20070135370, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Exemplary Influenza virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins(NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), andhaemagglutinin (HA). Influenza A NP sequences are set forth in, e.g.,Genbank Accession Nos. NC_004522; AY818138; AB166863; AB188817;AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493;AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651500;AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507;AY651509; AY651528; AY770996; AY790308; AY818138; and AY818140.Influenza A PA sequences are set forth in, e.g., Genbank Accession Nos.AY818132; AY790280; AY646171; AY818132; AY818133; AY646179; AY818134;AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611;AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615;AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995;and AY724786. Non-limiting examples of siRNA molecules targetingInfluenza virus nucleic acid sequences include those described in U.S.Patent Publication No. 20070218122, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Exemplary hepatitis virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences involved intranscription and translation (e.g., En1, En2, X, P) and nucleic acidsequences encoding structural proteins (e.g., core proteins including Cand C-related proteins, capsid and envelope proteins including S, M,and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY,supra). Exemplary Hepatitis C virus (HCV) nucleic acid sequences thatcan be silenced include, but are not limited to, the 5′-untranslatedregion (5′-UTR), the 3′-untranslated region (3′-UTR), the polyproteintranslation initiation codon region, the internal ribosome entry site(IRES) sequence, and/or nucleic acid sequences encoding the coreprotein, the E1 protein, the E2 protein, the p7 protein, the NS2protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein,the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase. HCVgenome sequences are set forth in, e.g., Genbank Accession Nos.NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCVgenotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4),NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). Hepatitis Avirus nucleic acid sequences are set forth in, e.g., Genbank AccessionNo. NC_001489; Hepatitis B virus nucleic acid sequences are set forthin, e.g., Genbank Accession No. NC_003977; Hepatitis D virus nucleicacid sequence are set forth in, e.g., Genbank Accession No. NC_001653;Hepatitis E virus nucleic acid sequences are set forth in, e.g., GenbankAccession No. NC_001434; and Hepatitis G virus nucleic acid sequencesare set forth in, e.g., Genbank Accession No. NC-001710. Silencing ofsequences that encode genes associated with viral infection and survivalcan conveniently be used in combination with the administration ofconventional agents used to treat the viral condition. Non-limitingexamples of siRNA molecules targeting hepatitis virus nucleic acidsequences include those described in U.S. Patent Publication Nos.20060281175, 20050058982, and 20070149470; U.S. Pat. No. 7,348,314; andU.S. Provisional Application No. 61/162,127, filed Mar. 20, 2009, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, for example, genes expressed in dyslipidemia (e.g., liver Xreceptors such as LXRα and LXRβ (Genback Accession No. NM_007121),farnesoid X receptors (FXR) (Genbank Accession No. NM_005123),sterol-regulatory element binding protein (SREBP), site-1 protease(SIP), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-Areductase), apolipoprotein B (ApoB) (Genbank Accession No. NM_000384),apolipoprotein CIII (ApoC3) (Genbank Accession Nos. NM_000040 andNG_008949 REGION: 5001.8164), and apolipoprotein E (ApoE) (GenbankAccession Nos. NM_000041 and NG-007084 REGION: 5001.8612)); and diabetes(e.g., glucose 6-phosphatase) (see, e.g., Forman et al., Cell, 81:687(1995); Seol et al., Mol. Endocrinol., 9:72 (1995), Zavacki et al.,Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell,85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272:12778-12785(1997); Willy et al., Genes Dev., 9:1033-1045 (1995); Lehmann et al., J.Biol. Chem., 272:3137-3140 (1997); Janowski et al., Nature, 383:728-731(1996); and Peet et al., Cell, 93:693-704 (1998)). One of skill in theart will appreciate that genes associated with metabolic diseases anddisorders (e.g., diseases and disorders in which the liver is a targetand liver diseases and disorders) include genes that are expressed inthe liver itself as well as and genes expressed in other organs andtissues. Silencing of sequences that encode genes associated withmetabolic diseases and disorders can conveniently be used in combinationwith the administration of conventional agents used to treat the diseaseor disorder. Non-limiting examples of siRNA molecules targeting the ApoBgene include those described in U.S. Patent Publication No. 20060134189,the disclosure of which is herein incorporated by reference in itsentirety for all purposes. Non-limiting examples of siRNA moleculestargeting the ApoC3 gene include those described in U.S. ProvisionalApplication No. 61/147,235, filed Jan. 26, 2009, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

Examples of gene sequences associated with tumorigenesis and celltransformation (e.g., cancer or other neoplasia) include mitotickinesins such as Eg5 (KSP, KIF11; Genbank Accession No. NM_004523);serine/threonine kinases such as polo-like kinase 1 (PLK-1) (GenbankAccession No. NM_005030; Barr et al., Nat. Rev. Mol. Cell. Biol.,5:429-440 (2004)); tyrosine kinases such as WEE1 (Genbank Accession Nos.NM_003390 and NM_001143976); inhibitors of apoptosis such as XIAP(Genbank Accession No. NM_001167); COP9 signalosome subunits such asCSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank Accession No. NM_006837);CSN6, CSN7A, CSN7B, and CSN8; ubiquitin ligases such as COP1 (RFWD2;Genbank Accession Nos. NM_022457 and NM_001001740); and histonedeacetylases such as HDAC1, HDAC2 (Genbank Accession No. NM_001527),HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. Non-limitingexamples of siRNA molecules targeting the Eg5 and XIAP genes includethose described in U.S. patent application Ser. No. 11/807,872, filedMay 29, 2007, the disclosure of which is herein incorporated byreference in its entirety for all purposes. Non-limiting examples ofsiRNA molecules targeting the PLK-1 gene include those described in U.S.Patent Publication Nos. 20050107316 and 20070265438; and U.S. patentapplication Ser. No. 12/343,342, filed Dec. 23, 2008, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. Non-limiting examples of siRNA molecules targeting the CSN5gene include those described in U.S. Provisional Application No.61/045,251, filed Apr. 15, 2008, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Additional examples of gene sequences associated with tumorigenesis andcell transformation include translocation sequences such as MLL fusiongenes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al.,Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2,AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003));overexpressed sequences such as multidrug resistance genes (Nieth etal., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res. 63:1515 (2003)),cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev.,16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase genes (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR/ErbB1(Genbank Accession Nos. NM_005228, NM_201282, NM_201283, and NM_201284;see also, Nagy et al. Exp. Cell Res., 285:39-49 (2003), ErbB2/HER-2(Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (GenbankAccession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank AccessionNos. NM_005235 and NM_001042599); and mutated sequences such as RAS(reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)).Non-limiting examples of siRNA molecules targeting the EGFR gene includethose described in U.S. patent application Ser. No. 11/807,872, filedMay 29, 2007, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

Silencing of sequences that encode DNA repair enzymes find use incombination with the administration of chemotherapeutic agents (Colliset al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associatedwith tumor migration are also target sequences of interest, for example,integrins, selectins, and metalloproteinases. The foregoing examples arenot exclusive. Those of skill in the art will understand that any wholeor partial gene sequence that facilitates or promotes tumorigenesis orcell transformation, tumor growth, or tumor migration can be included asa template sequence.

Angiogenic genes are able to promote the formation of new vessels. Ofparticular interest is vascular endothelial growth factor (VEGF) (Reichet al., Mol. Vis., 9:210 (2003)) or VEGFR. siRNA sequences that targetVEGFR are set forth in, e.g., GB 2396864; U.S. Patent Publication No.20040142895; and CA 2456444, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Anti-angiogenic genes are able to inhibit neovascularization. Thesegenes are particularly useful for treating those cancers in whichangiogenesis plays a role in the pathological development of thedisease. Examples of anti-angiogenic genes include, but are not limitedto, endostatin (see, e.g., U.S. Pat. No. 6,174,861), angiostatin (see,e.g., U U.S. Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin etal., J. Pathol., 188: 369-377 (1999)), the disclosures of which areherein incorporated by reference in their entirety for all purposes.Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include, withoutlimitation, cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF,FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2,IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18,IL-20, etc.), interferons (e.g., IFN-α, IFN—P, IFN-γ, etc.) and TNF. Fasand Fas ligand genes are also immunomodulator target sequences ofinterest (Song et al., Nat. Med., 9:347 (2003)). Genes encodingsecondary signaling molecules in hematopoietic and lymphoid cells arealso included in the present invention, for example, Tec family kinasessuch as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett.,527:274 (2002)).

Cell receptor ligands include ligands that are able to bind to cellsurface receptors (e.g., insulin receptor, EPO receptor, G-proteincoupled receptors, receptors with tyrosine kinase activity, cytokinereceptors, growth factor receptors, etc.), to modulate (e.g., inhibit,activate, etc.) the physiological pathway that the receptor is involvedin (e.g., glucose level modulation, blood cell development, mitogenesis,etc.). Examples of cell receptor ligands include, but are not limitedto, cytokines, growth factors, interleukins, interferons, erythropoietin(EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.Templates coding for an expansion of trinucleotide repeats (e.g., CAGrepeats) find use in silencing pathogenic sequences in neurodegenerativedisorders caused by the expansion of trinucleotide repeats, such asspinobulbular muscular atrophy and Huntington's Disease (Caplen et al.,Hum. Mol. Genet., 11:175 (2002)).

Certain other target genes, which may be targeted by a nucleic acid(e.g., by siRNA) to downregulate or silence the expression of the gene,include but are not limited to, Actin, Alpha 2, Smooth Muscle, Aorta(ACTA2), Alcohol dehydrogenase 1A (ADH1A), Alcohol dehydrogenase 4(ADH4), Alcohol dehydrogenase 6 (ADH6), Afamin (AFM), Angiotensinogen(AGT), Serine-pyruvate aminotransferase (AGXT), Alpha-2-HS-glycoprotein(AHSG), Aldo-keto reductase family 1 member C4 (AKR1C4), Serum albumin(ALB), alpha-1-microglobulin/bikunin precursor (AMBP),Angiopoietin-related protein 3 (ANGPTL3), Serum amyloid P-component(APCS), Apolipoprotein A-II (APOA2), Apolipoprotein B-100 (APOB),Apolipoprotein C3 (APOC3), Apolipoprotein C-IV (APOC4), Apolipoprotein F(APOF), Beta-2-glycoprotein 1 (APOH), Aquaporin-9 (AQP9), Bileacid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein betachain (C4BPB), Putative uncharacterized protein encoded by LINC01554(C5orf27), Complement factor 3 (C3), Complement Factor 5 (C5),Complement component C6 (C6), Complement component C8 alpha chain (C8A),Complement component C8 beta chain (C8B), Complement component C8 gammachain (C8G), Complement component C9 (C9), Calmodulin BindingTranscription Activator 1 (CAMTA1), CD38 (CD38), Complement Factor B(CFB), Complement factor H-related protein 1 (CFHR1), Complement factorH-related protein 2 (CFHR2), Complement factor H-related protein 3(CFHR3), Cannabinoid receptor 1 (CNR1), ceruloplasmin (CP),carboxypeptidase B2 (CPB2), Connective tissue growth factor (CTGF),C-X-C motif chemokine 2 (CXCL2), Cytochrome P450 1A2 (CYP1A2),Cytochrome P450 2A6 (CYP2A6), Cytochrome P450 2C8 (CYP2C8), CytochromeP450 2C9 (CYP2C9), Cytochrome P450 Family 2 Subfamily D Member 6(CYP2D6), Cytochrome P450 2E1 (CYP2E1), Phylloquinone omega-hydroxylaseCYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase(CYP8B1), Dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12),coagulation factor II (thrombin) (F2), coagulation factor IX (F9),fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), fibrinogengamma chain (FGG), fibrinogen-like 1 (FGL1), flavin containingmonooxygenase 3 (FMO3), flavin containing monooxygenase 5 (FMO5),group-specific component (vitamin D binding protein) (GC), Growthhormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronanbinding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP),hydroxyacid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC),haptoglobin-related protein; haptoglobin (HPR), hemopexin (HPX),histidine-rich glycoprotein (HRG), hydroxysteroid (11-beta)dehydrogenase 1 (HSDIIB1), hydroxysteroid (17-beta) dehydrogenase 13(HSD17B13), Inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1),Inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2),Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3),Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Prekallikrein(KLKB1), Lactate dehydrogenase A (LDHA), liver expressed antimicrobialpeptide 2 (LEAP2), leukocyte cell-derived chemotaxin 2 (LECT2),Lipoprotein (a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2),S-adenosylmethionine synthase isoform type-1 (MAT1A), NADPH Oxidase 4(NOX4), Poly [ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1),paraoxonase 3 (PON3), Vitamin K-dependent protein C (PROC), Retinoldehydrogenase 16 (RDH16), serum amyloid A4, constitutive (SAA4), serinedehydratase (SDS), Serpin Family A Member 1 (SERPINA1), Serpin A11(SERPINA11), Kallistatin (SERPINA4), Corticosteroid-binding globulin(SERPINA6), Antithrombin-III (SERPINC1), Heparin cofactor 2 (SERPIND1),Serpin Family H Member 1 (SERPINH1), Solute Carrier Family 5 Member 2(SLC5A2), Sodium/bile acid cotransporter (SLC10A1), Solute carrierfamily 13 member 5 (SLC13A5), Solute carrier family 22 member 1(SLC22A1), Solute carrier family 25 member 47 (SLC25A47), Solute carrierfamily 2, facilitated glucose transporter member 2 (SLC2A2),Sodium-coupled neutral amino acid transporter 4 (SLC38A4), Solutecarrier organic anion transporter family member 1B1 (SLCO1B1),Sphingomyelin Phosphodiesterase 1 (SMPD1), Bile salt sulfotransferase(SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase(TDO2), UDP glucuronosyltransferase 2 family, polypeptide B10 (UGT2B10),UDP glucuronosyltransferase 2 family, polypeptide B15 (UGT2B15), UDPglucuronosyltransferase 2 family, polypeptide B4 (UGT2B4) andvitronectin (VTN).

In addition to its utility in silencing the expression of any of theabove-described genes for therapeutic purposes, certain nucleic acids(e.g., siRNA) described herein are also useful in research anddevelopment applications as well as diagnostic, prophylactic,prognostic, clinical, and other healthcare applications. As anon-limiting example, certain nucleic acids (e.g., siRNA) can be used intarget validation studies directed at testing whether a gene of interesthas the potential to be a therapeutic target. Certain nucleic acids(e.g., siRNA) can also be used in target identification studies aimed atdiscovering genes as potential therapeutic targets.

CRISPR

Targeted genome editing has progressed from being a niche technology toa method used by many biological researchers. This progression has beenlargely fueled by the emergence of the clustered, regularly interspaced,short palindromic repeat (CRISPR) technology (see, e.g., Sander et al.,Nature Biotechnology, 32(4), 347-355, including SupplementaryInformation (2014) and International Publication Numbers WO 2016/197132and WO 2016/197133). Accordingly, provided herein are improvements(e.g., lipid nanoparticles and formulations thereof) that can be used incombination with CRISPR technology to treat diseases, such as HBV.Regarding the targets for using CRISPR, the guide RNA (gRNA) utilized inthe CRISPR technology can be designed to target specifically identifiedsequences, e.g., target genes, e.g., of the HBV genome. Examples of suchtarget sequences are provided in International Publication Number WO2016/197132. Further, International Publication Number WO 2013/151665(e.g., see Table 6; which document is specifically incorporated byreference, particularly including Table 6, and the associated SequenceListing) describes about 35,000 mRNA sequences, claimed in the contextof an mRNA expression construct. Certain embodiments of the presentinvention utilize CRISPR technology to target the expression of any ofthese sequences. Certain embodiments of the present invention may alsoutilize CRISPR technology to target the expression of a target genediscussed herein.

aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is preferably about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”,etc.). In certain aspects, the aiRNA molecules described herein maycomprise one or more modified nucleotides, e.g., in the double-stranded(duplex) region and/or in the antisense overhangs. As a non-limitingexample, aiRNA sequences may comprise one or more of the modifiednucleotides described above for siRNA sequences. In a preferredembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein. In other embodiments, aiRNAmolecules may be used to silence the expression of any of the targetgenes set forth above, such as, e.g., genes associated with viralinfection and survival, genes associated with metabolic diseases anddisorders, genes associated with tumorigenesis and cell transformation,angiogenic genes, immunomodulator genes such as those associated withinflammatory and autoimmune responses, ligand receptor genes, and genesassociated with neurodegenerative disorders.

miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294:862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule. miRNA are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,^(˜)70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are preferably about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In a preferred embodiment, the miRNA molecule comprises2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In some embodiments, miRNA molecules may be used to silence theexpression of any of the target genes set forth above, such as, e.g.,genes associated with viral infection and survival, genes associatedwith metabolic diseases and disorders, genes associated withtumorigenesis and cell transformation, angiogenic genes, immunomodulatorgenes such as those associated with inflammatory and autoimmuneresponses, ligand receptor genes, and genes associated withneurodegenerative disorders.

In other embodiments, one or more agents that block the activity of amiRNA targeting an mRNA of interest are administered using a lipidparticle of the invention (e.g., a nucleic acid-lipid particle).Examples of blocking agents include, but are not limited to, stericblocking oligonucleotides, locked nucleic acid oligonucleotides, andMorpholino oligonucleotides. Such blocking agents may bind directly tothe miRNA or to the miRNA binding site on the target mRNA.

Antisense Oligonucleotides

In one embodiment, the nucleic acid is an antisense oligonucleotidedirected to a target gene or sequence of interest. The terms “antisenseoligonucleotide” or “antisense” include oligonucleotides that arecomplementary to a targeted polynucleotide sequence. Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Antisense RNA oligonucleotides prevent thetranslation of complementary RNA strands by binding to the RNA.Antisense DNA oligonucleotides can be used to target a specific,complementary (coding or non-coding) RNA. If binding occurs, thisDNA/RNA hybrid can be degraded by the enzyme RNase H. In a particularembodiment, antisense oligonucleotides comprise from about 10 to about60 nucleotides, more preferably from about 15 to about 30 nucleotides.The term also encompasses antisense oligonucleotides that may not beexactly complementary to the desired target gene. Thus, the inventioncan be utilized in instances where non-target specific-activities arefound with antisense, or where an antisense sequence containing one ormore mismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(see, U.S. Pat. Nos. 5,739,119 and 5,759,829). Furthermore, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDR1), ICAM-1, E-selectin,STK-1, striatal GABAA receptor, and human EGF (see, Jaskulski et al.,Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun.,1:225-32 (1989); Penis et al., Brain Res Mol Brain Res., 15; 57:310-20(1998); and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288). Moreover, antisense constructs have also been described thatinhibit and can be used to treat a variety of abnormal cellularproliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317;and 5,783,683). The disclosures of these references are hereinincorporated by reference in their entirety for all purposes.

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

Ribozymes According to another embodiment of the invention, nucleicacid-lipid particles are associated with ribozymes. Ribozymes areRNA-protein complexes having specific catalytic domains that possessendonuclease activity (see, Kim et al., Proc. Natl. Acad. Sci. USA.,84:8788-92 (1987); and Forster et al., Cell, 49:211-20 (1987)). Forexample, a large number of ribozymes accelerate phosphoester transferreactions with a high degree of specificity, often cleaving only one ofseveral phosphoesters in an oligonucleotide substrate (see, Cech et al.,Cell, 27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610(1990); Reinhold-Hurek et al., Nature, 357:173-6 (1992)). Thisspecificity has been attributed to the requirement that the substratebind via specific base-pairing interactions to the internal guidesequence (“IGS”) of the ribozyme prior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAmolecules are known presently. Each can catalyze the hydrolysis of RNAphosphodiester bonds in trans (and thus can cleave other RNA molecules)under physiological conditions. In general, enzymatic nucleic acids actby first binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, hepatitis δ virus, group I intron or RNaseP RNA (in associationwith an RNA guide sequence), or Neurospora VS RNA motif, for example.Specific examples of hammerhead motifs are described in, e.g., Rossi etal., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifsare described in, e.g., EP 0360257, Hampel et al., Biochemistry,28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990);and U.S. Pat. No. 5,631,359. An example of the hepatitis δ virus motifis described in, e.g., Perrotta et al., Biochemistry, 31:11843-52(1992). An example of the RNaseP motif is described in, e.g.,Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of theNeurospora VS RNA ribozyme motif is described in, e.g., Saville et al.,Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA,88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Anexample of the Group I intron is described in, e.g., U.S. Pat. No.4,987,071. Important characteristics of enzymatic nucleic acid moleculesused according to the invention are that they have a specific substratebinding site which is complementary to one or more of the target geneDNA or RNA regions, and that they have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. Thus, the ribozyme constructs need not belimited to specific motifs mentioned herein. The disclosures of thesereferences are herein incorporated by reference in their entirety forall purposes.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in, e.g.,PCT Publication Nos. WO 93/23569 and WO 94/02595, and synthesized to betested in vitro and/or in vivo as described therein. The disclosures ofthese PCT publications are herein incorporated by reference in theirentirety for all purposes.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases(see, e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO 91/03162,and WO 94/13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, whichdescribe various chemical modifications that can be made to the sugarmoieties of enzymatic RNA molecules, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes),modifications which enhance their efficacy in cells, and removal of stemII bases to shorten RNA synthesis times and reduce chemicalrequirements.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal such as a human.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see, Yamamoto et al., J. Immunol., 148:4072-6 (1992)), orCpG motifs, as well as other known ISS features (such as multi-Gdomains; see; PCT Publication No. WO 96/11266, the disclosure of whichis herein incorporated by reference in its entirety for all purposes).

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target sequence in order to provoke an immuneresponse. Thus, certain immunostimulatory nucleic acids may comprise asequence corresponding to a region of a naturally-occurring gene ormRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein the CpG dinucleotide is methylated. In an alternative embodiment, thenucleic acid comprises at least two CpG dinucleotides, wherein at leastone cytosine in the CpG dinucleotides is methylated. In a furtherembodiment, each cytosine in the CpG dinucleotides present in thesequence is methylated. In another embodiment, the nucleic acidcomprises a plurality of CpG dinucleotides, wherein at least one of theCpG dinucleotides comprises a methylated cytosine. Examples ofimmunostimulatory oligonucleotides suitable for use in the compositionsand methods of the present invention are described in PCT ApplicationNo. PCT/US08/88676, filed Dec. 31, 2008, PCT Publication Nos.

WO 02/069369 and WO 01/15726, U.S. Pat. No. 6,406,705, and Raney et al.,J. Pharm. Exper. Ther., 298:1185-92 (2001), the disclosures of which areeach herein incorporated by reference in their entirety for allpurposes. In certain embodiments, the oligonucleotides used in thecompositions and methods of the invention have a phosphodiester (“PO”)backbone or a phosphorothioate (“PS”) backbone, and/or at least onemethylated cytosine residue in a CpG motif.

mRNA

In certain embodiments, the nucleic acid is one or more mRNA molecules(e.g, a cocktail of mRNA molecules).

Modifications to mRNA

mRNA used in the practice of the present invention can include one, two,or more than two nucleoside modifications. In some embodiments, themodified mRNA exhibits reduced degradation in a cell into which the mRNAis introduced, relative to a corresponding unmodified mRNA.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methy1-pseudouridine, 4-thio-1-methy 1-pseudouridine, 2-thio-1-methy1-pseudouridine, 1-methy 1-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihy drouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In specific embodiments, a modified nucleoside is5′-0-(1-Thiophosphate)-Adenosine, 5′-0-(1-Thiophosphate)-Cytidine,5′-0-(1-Thiophosphate)-Guanosine, 5′-0-(1-Thiophosphate)-Uridine or5′-0-(1-Thiophosphate)-Pseudouridine. The α-thio substituted phosphatemoiety is provided to confer stability to RNA polymers through theunnatural phosphorothioate backbone linkages. Phosphorothioate RNA haveincreased nuclease resistance and subsequently a longer half-life in acellular environment. Phosphorothioate linked nucleic acids are expectedto also reduce the innate immune response through weakerbinding/activation of cellular innate immune molecules.

In certain embodiments it is desirable to intracellularly degrade amodified nucleic acid introduced into the cell, for example if precisetiming of protein production is desired. Thus, the invention provides amodified nucleic acid containing a degradation domain, which is capableof being acted on in a directed manner within a cell.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

Optional Components of the Modified Nucleic Acids

In further embodiments, the modified nucleic acids may include otheroptional components, which can be beneficial in some embodiments. Theseoptional components include, but are not limited to, untranslatedregions, kozak sequences, intronic nucleotide sequences, internalribosome entry site (IRES), caps and polyA tails. For example, a 5′untranslated region (UTR) and/or a 3′ UTR may be provided, whereineither or both may independently contain one or more differentnucleoside modifications. In such embodiments, nucleoside modificationsmay also be present in the translatable region. Also provided arenucleic acids containing a Kozak sequence.

Additionally, provided are nucleic acids containing one or more intronicnucleotide sequences capable of being excised from the nucleic acid.

Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into the mRNA used inthe present invention to increase the stability of the molecule. Thespecific features can also be incorporated to ensure controlleddown-regulation of the transcript in case they are misdirected toundesired organs sites.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

IRES Sequences

mRNA containing an internal ribosome entry site (TRES) are also usefulin the practice of the present invention. An IRES may act as the soleribosome binding site, or may serve as one of multiple ribosome bindingsites of an mRNA. An mRNA containing more than one functional ribosomebinding site may encode several peptides or polypeptides that aretranslated independently by the ribosomes (“multicistronic mRNA”). WhenmRNA are provided with an TRES, further optionally provided is a secondtranslatable region. Examples of RES sequences that can be usedaccording to the invention include without limitation, those frompicornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecules in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between 100 and 250residues long.

Generally, the length of a poly-A tail is greater than 30 nucleotides inlength. In another embodiment, the poly-A tail is greater than 35nucleotides in length (e.g., at least or greater than about 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides).

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the modified mRNA. The poly-A tailmay also be designed as a fraction of modified nucleic acids to which itbelongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60,70, 80, or 90% or more of the total length of the modified mRNA or thetotal length of the modified mRNA minus the poly-A tail.

Generating mRNA Molecules

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989));as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202; PCRProtocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Expression libraries are also well known to those of skill inthe art. Additional basic texts disclosing the general methods of use inthis invention include Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994). The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

Encoded Polypeptides

The mRNA component of a nucleic acid-lipid particle described herein canbe used to express a polypeptide of interest. Certain diseases in humansare caused by the absence or impairment of a functional protein in acell type where the protein is normally present and active. Thefunctional protein can be completely or partially absent due, e.g., totranscriptional inactivity of the encoding gene or due to the presenceof a mutation in the encoding gene that renders the protein completelyor partially non-functional. Examples of human diseases that are causedby complete or partial inactivation of a protein include X-linked severecombined immunodeficiency (X-SCID) and adrenoleukodystrophy (X-ALD).X-SCID is caused by one or more mutations in the gene encoding thecommon gamma chain protein that is a component of the receptors forseveral interleukins that are involved in the development and maturationof B and T cells within the immune system. X-ALD is caused by one ormore mutations in a peroxisomal membrane transporter protein gene calledABCD1. Individuals afflicted with X-ALD have very high levels of longchain fatty acids in tissues throughout the body, which causes a varietyof symptoms that may lead to mental impairment or death.

Attempts have been made to use gene therapy to treat some diseasescaused by the absence or impairment of a functional protein in a celltype where the protein is normally present and active. Gene therapytypically involves introduction of a vector that includes a geneencoding a functional form of the affected protein, into a diseasedperson, and expression of the functional protein to treat the disease.Thus far, gene therapy has met with limited success. Additionally,certain aspects of delivering mRNA using LNPs have been described, e.g.,in International Publication Numbers WO 2018/006052 and WO 2015/011633.

As such, there is a continuing need for improvement for expressing afunctional form of a protein within a human who suffers from a diseasecaused by the complete or partial absence of the functional protein, andthere is a need for improved delivery of nucleic acids (e.g., mRNA) viaa methods and compositions, e.g., that can trigger less of an immuneresponse to the therapy. Certain embodiments of the present inventionare useful in this context. Thus, in certain embodiments, expression ofthe polypeptide ameliorates one or more symptoms of a disease ordisorder. Certain compositions and methods of the invention may beuseful for treating human diseases caused by the absence, or reducedlevels, of a functional polypeptide within the human body. In otherembodiments, certain compositions and methods of the invention may beuseful for expressing a vaccine antigen for treating cancer.

Self-Amplifying RNA

In certain embodiments, the nucleic acid is one or more self-amplifyingRNA molecules. Self-amplifying RNA (sa-RNA) may also be referred to asself-replicating RNA, replication-competent RNA, replicons or RepRNA.RepRNA, referred to as self-amplifying mRNA when derived frompositive-strand viruses, is generated from a viral genome lacking atleast one structural gene; it can translate and replicate (hence“self-amplifying”) without generating infectious progeny virus. Incertain embodiments, the RepRNA technology may be used to insert a genecassette encoding a desired antigen of interest. For example, thealphaviral genome is divided into two open reading frames (ORFs): thefirst ORF encodes proteins for the RNA dependent RNA polymerase(replicase), and the second ORF encodes structural proteins. In sa-RNAvaccine constructs, the ORF encoding viral structural proteins may bereplaced with any antigen of choice, while the viral replicase remainsan integral part of the vaccine and drives intracellular amplificationof the RNA after immunization.

Other Active Agents

In certain embodiments, the active agent associated with the lipidparticles of the invention may comprise one or more therapeuticproteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugsinclude oncology drugs (e.g., chemotherapy drugs, hormonal therapeuticagents, immunotherapeutic agents, radiotherapeutic agents, etc.),lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds,antidepressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs such asanti-arrhythmic agents, hormones, vasoconstrictors, and steroids. Theseactive agents may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising nucleic acid, such as interferingRNA or mRNA.

Non-limiting examples of chemotherapy drugs include platinum-based drugs(e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin,satraplatin, etc.), alkylating agents (e.g., cyclophosphamide,ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine,uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g.,5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel(taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan(CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g.,gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®;OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib(SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib(Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101),vandetanib (Zactima™; ZD6474), etc.), pharmaceutically acceptable saltsthereof, stereoisomers thereof, derivatives thereof, analogs thereof,and combinations thereof.

Examples of conventional hormonal therapeutic agents include, withoutlimitation, steroids (e.g., dexamethasone), finasteride, aromataseinhibitors, tamoxifen, and goserelin as well as othergonadotropin-releasing hormone agonists (GnRH).

Examples of conventional immunotherapeutic agents include, but are notlimited to, immunostimulants (e.g., Bacillus Calmette-Guerin (BCG),levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.).

Examples of conventional radiotherapeutic agents include, but are notlimited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷, Y⁹⁰,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

Additional oncology drugs that may be used according to the inventioninclude, but are not limited to, alkeran, allopurinol, altretamine,amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU,carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosinearabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane,FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin,interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol,L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate,Pegademase, pentostatin, porfimer sodium, prednisone, rituxan,streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene,tretinoin, ATRA, valrubicin, and velban. Other examples of oncologydrugs that may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors, and camptothecins.

Non-limiting examples of lipid-lowering agents for treating a lipiddisease or disorder associated with elevated triglycerides, cholesterol,and/or glucose include statins, fibrates, ezetimibe, thiazolidinediones,niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, andmixtures thereof.

Examples of anti-viral drugs include, but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol,atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, entry inhibitors, famciclovir, fixed dose combinations,fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors,ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitors, interferon type III (e.g., IFN-Xmolecules such as IFN-k1, IFN-λ2, and IFN-λ3), interferon type II (e.g.,IFN-γ), interferon type I (e.g., IFN-α such as PEGylated IFN-α, IFN-β,IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ, interferon, lamivudine,lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir,peramivir, pleconaril, podophyllotoxin, protease inhibitors, reversetranscriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir,stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, zidovudine, pharmaceutically acceptable salts thereof,stereoisomers thereof, derivatives thereof, analogs thereof, andmixtures thereof.

Lipid Particles

The lipid particles of the invention typically comprise an active agentor therapeutic agent, a cationic lipid, a non-cationic lipid, and aconjugated lipid that inhibits aggregation of particles. In someembodiments, the active agent or therapeutic agent is fully encapsulatedwithin the lipid portion of the lipid particle such that the activeagent or therapeutic agent in the lipid particle is resistant in aqueoussolution to enzymatic degradation, e.g., by a nuclease or protease. Inother embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 40 nm toabout 150 nm, from about 50 nm to about 150 nm, from about 60 nm toabout 130 nm, from about 70 nm to about 110 nm, or from about 70 toabout 90 nm.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (LNP) which comprise one ormore nucleic acid molecules, such as an interfering RNA (e.g., siRNA,aiRNA, and/or miRNA) or mRNA; a cationic lipid (e.g., a cationic lipidof Formulas I, II, and/or III); a non-cationic lipid (e.g., cholesterolalone or mixtures of one or more phospholipids and cholesterol); and aconjugated lipid that inhibits aggregation of the particles (e.g., oneor more PEG-lipid conjugates). The LNP may comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more unmodified and/or modified nucleic acidmolecules. Nucleic acid-lipid particles and their method of preparationare described in, e.g., U.S. Pat. Nos. 5,753,613; 5,785,992; 5,705,385;5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication No.WO 96/40964, the disclosures of which are each herein incorporated byreference in their entirety for all purposes.

Cationic Lipids

In the lipid nanoparticles of the invention, the cationic lipid can beselected from compounds of formula (I):

wherein:

R¹ is a C₂-C₃₀ hydrocarbyl;

R² is a C₂-C₃₀ hydrocarbyl;

R³ is a C₂-C₃₀ hydrocarbyl;

X is a divalent C₂-C₈ alkyl;

R⁴ is NR^(a)R^(b); and

each R^(a) and R^(b) is independently selected from the group consistingof methyl, ethyl, propyl, cyclopropyl, and butyl, which methyl, ethyl,propyl, cyclopropyl, and butyl is optionally substituted with hydroxy;or R^(a) and R^(b) taken with the nitrogen to which they are attachedform an aziridine, azetidine, proline, piperidine, piperazine, ormorpholine ring, which ring is optionally substituted with hydroxyl orwith C₁-C₆ alkyl that is optionally substituted with hydroxy.

In one embodiment R¹ is a C₂-C₂₀ hydrocarbyl.

In one embodiment R¹ is a C₂-C₁₅ hydrocarbyl.

In one embodiment R¹ is a C₂-C₁₀ hydrocarbyl.

In one embodiment R¹ is a C₅-C₂₀ hydrocarbyl.

In one embodiment R¹ is a (C₂-C₂₀)alkyl, (C₂-C₂₀)alkenyl, or(C₂-C₂₀)alkynyl

In one embodiment R¹ is a (C₈-C₂₀)alkyl.

In one embodiment R¹ is a (C₈-C₂₀)alkenyl.

In one embodiment R¹ is a (C₈-C₂₀)alkynyl.

In one embodiment R¹ is a (C₈-C₂₀)alkenyl, having only one double bond.

In one embodiment R¹ is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl,6(Z), 15(Z)-henicosandiene-11-yl, 3-hexen-1-yl, 9(Z)-octadecene-1-yl, or2-butyloct-1-yl, 4-(1-methylethenyl)cyclohexen-1-ylmethyl.

In one embodiment R² is a C₂-C₂₀ hydrocarbyl.

In one embodiment R² is a C₂-C₁₅ hydrocarbyl.

In one embodiment R² is a C₂-C₁₀ hydrocarbyl.

In one embodiment R² is a C₅-C₂₀ hydrocarbyl.

In one embodiment R² is a (C₂-C₂₀)alkyl, (C₂-C₂₀)alkenyl, or(C₂-C₂₀)alkynyl

In one embodiment R² is a (C₈-C₂₀)alkyl.

In one embodiment R² is a (C₈-C₂₀)alkenyl.

In one embodiment R² is a (C₈-C₂₀)alkynyl.

In one embodiment R² is a (C₈-C₂₀)alkenyl, having only one double bond.

In one embodiment R² is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl,6(Z), 15(Z)-henicosandiene-11-yl, 3-hexen-1-yl, 9(Z)-octadecene-1-yl, or2-butyloct-1-yl, 4-(1-methylethenyl)cyclohexen-1-ylmethyl.

In one embodiment R³ is a C₂-C₂₀ hydrocarbyl.

In one embodiment R³ is a C₂-C₁₅ hydrocarbyl.

In one embodiment R³ is a C₂-C₁₀ hydrocarbyl.

In one embodiment R³ is a C₅-C₂₀ hydrocarbyl.

In one embodiment R³ is a (C₂-C₂₀)alkyl, (C₂-C₂₀)alkenyl, or(C₂-C₂₀)alkynyl

In one embodiment R³ is a (C₈-C₂₀)alkyl.

In one embodiment R³ is a (C₈-C₂₀)alkenyl.

In one embodiment R³ is a (C₈-C₂₀)alkynyl.

In one embodiment R³ is a (C₈-C₂₀)alkenyl, having only one double bond.

In one embodiment R³ is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl,6(Z), 15(Z)-henicosandiene-11-yl, 3-hexen-1-yl, 9(Z)-octadecene-1-yl,adamantly-1-ylmethyl, 2-butyloct-1-yl,(Z)-2-((Z)-dec-4-en-1-yl)dodec-6-en-1-yl, or4-(prop-1-ene-2-yl)cyclohex-1-en-1-yl)methyl.

In one embodiment X is a divalent C₂-C₆ alkyl.

In one embodiment X is a divalent C₃-C₅ alkyl.

In one embodiment X is —CH₂CH₂CH₂—.

In one embodiment X is —CH₂CH₂CH₂CH₂—.

In one embodiment X is —CH₂CH₂CH₂ CH₂CH₂—.

In one embodiment each R^(a) and R^(b) is independently selected fromthe group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl,which methyl, ethyl, propyl, cyclopropyl, and butyl is optionallysubstituted with hydroxy.

In one embodiment R^(a) and R^(b) taken with the nitrogen to which theyare attached form an aziridine, azetidine, proline, piperidine,piperazine, or morpholine ring, which ring is optionally substitutedwith hydroxyl or with C₁-C₆ alkyl that is optionally substituted withhydroxy.

In one embodiment each R^(a) and R^(b) is independently selected fromthe group consisting of methyl and ethyl.

In one embodiment R⁴ is dimethylamino.

Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., LNP) can be any of a variety of neutral uncharged, zwitterionic,or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., LNP) comprises or consists of cholesterol or aderivative thereof, e.g., a phospholipid-free lipid particleformulation. In other embodiments, the non-cationic lipid present in thelipid particles (e.g., LNP) comprises or consists of one or morephospholipids, e.g., a cholesterol-free lipid particle formulation. Infurther embodiments, the non-cationic lipid present in the lipidparticles (e.g., LNP) comprises or consists of a mixture of one or morephospholipids and cholesterol or a derivative thereof.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 13 mol% to about 49.5 mol %, from about 20 mol % to about 45 mol %, from about25 mol % to about 45 mol %, from about 30 mol % to about 45 mol %, fromabout 35 mol % to about 45 mol %, from about 20 mol % to about 40 mol %,from about 25 mol % to about 40 mol %, or from about 30 mol % to about40 mol % of the total lipid present in the particle.

In certain embodiments, the cholesterol present in phospholipid-freelipid particles comprises from about 30 mol % to about 45 mol %, fromabout 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %,or from about 35 mol % to about 40 mol % of the total lipid present inthe particle. As a non-limiting example, a phospholipid-free lipidparticle may comprise cholesterol at about 37 mol % of the total lipidpresent in the particle.

In certain other embodiments, the cholesterol present in lipid particlescontaining a mixture of phospholipid and cholesterol comprises fromabout 30 mol % to about 40 mol %, from about 30 mol % to about 35 mol %,or from about 35 mol % to about 40 mol % of the total lipid present inthe particle. As a non-limiting example, a lipid particle comprising amixture of phospholipid and cholesterol may comprise cholesterol atabout 34 mol % of the total lipid present in the particle.

In further embodiments, the cholesterol present in lipid particlescontaining a mixture of phospholipid and cholesterol comprises fromabout 10 mol % to about 30 mol %, from about 15 mol % to about 25 mol %,or from about 17 mol % to about 23 mol % of the total lipid present inthe particle. As a non-limiting example, a lipid particle comprising amixture of phospholipid and cholesterol may comprise cholesterol atabout 20 mol % of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40, 45, 50, 55, or 60 mol % of the total lipidpresent in the particle. In certain instances, the phospholipidcomponent in the mixture may comprise from about 2 mol % to about 12 mol%, from about 4 mol % to about 10 mol %, from about 5 mol % to about 10mol %, from about 5 mol % to about 9 mol %, or from about 6 mol % toabout 8 mol % of the total lipid present in the particle. As anon-limiting example, a lipid particle comprising a mixture ofphospholipid and cholesterol may comprise a phospholipid such as DPPC orDSPC at about 7 mol % (e.g., in a mixture with about 34 mol %cholesterol) of the total lipid present in the particle. In certainother instances, the phospholipid component in the mixture may comprisefrom about 10 mol % to about 30 mol %, from about 15 mol % to about 25mol %, or from about 17 mol % to about 23 mol % of the total lipidpresent in the particle. As another non-limiting example, a lipidparticle comprising a mixture of phospholipid and cholesterol maycomprise a phospholipid such as DPPC or DSPC at about 20 mol % (e.g., ina mixture with about 20 mol % cholesterol) of the total lipid present inthe particle.

Lipid Conjugate

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., LNP) comprise a lipid conjugate. The conjugatedlipid is useful in that it prevents the aggregation of particles.Suitable conjugated lipids include, but are not limited to, PEG-lipidconjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates(CPLs), and mixtures thereof. In certain embodiments, the particlescomprise either a PEG-lipid conjugate or an ATTA-lipid conjugatetogether with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes. AdditionalPEG-lipids include, without limitation, PEG-C-DOMG, 2 KPEG-DMG, and amixture thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). OtherPEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150(e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipidconjugates of the present invention. The disclosures of these patentsare herein incorporated by reference in their entirety for all purposes.In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CH₂COOH) isparticularly useful for preparing PEG-lipid conjugates including, e.g.,PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Additional PEG-lipid conjugates suitable for use in the inventioninclude, but are not limited to, compounds of formula:

A-B-C

or a salt thereof, wherein:

A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, or (C₂-C₆)alkanoyloxy, whereinany (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy issubstituted with one or more anionic precursor groups, and wherein any(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, hydroxyl, (C₁-C₃)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₁-C₃)alkylthio, or(C₂-C₃)alkanoyloxy;

B is a polyethylene glycol chain having a molecular weight of from about550 daltons to about 10,000 daltons;

C is -L-R^(a)

L is selected from the group consisting of a direct bond, —C(O)O—,—C(O)NR^(b)—, —NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—,—S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—;

R^(a) is a branched (C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl whereinone or more carbon atoms of the branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl have been replaced with —O—; and

each R^(b) is independently H or (C₁-C₆)alkyl.

The conjugated lipids may comprise a PEG-lipid including, e.g., acompound of formula A-PEG-diacylglycerol (DAG), A-PEG dialkyloxypropyl(DAA), A-PEG-phospholipid, A-PEG-ceramide (Cer), or mixtures thereof,wherein A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkylthio, or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy is substituted with one or moreanionic precursor groups, and wherein any (C₁-C₆)alkyl,(C₃-C₅)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy is optionally substituted withone or more groups independently selected from the group consisting ofhalo, hydroxyl, (C₁-C₃)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₁-C₃)alkylthio, or (C₂-C₃)alkanoyloxy. The A-PEG-DAA conjugate may beA-PEG-dilauryloxypropyl (C12), A-PEG-dimyristyloxypropyl (C14),A-PEG-dipalmityloxypropyl (C16), or A-PEG-distearyloxypropyl (C18), ormixtures thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “ATTR” or “polyamide” refers to, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” refers to a compound having 2 fatty acylchains, R¹ and R², both of which have independently between 2 and 30carbons bonded to the 1- and 2-position of glycerol by ester linkages.The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauryl (C₁₂), myristyl (C₁₄), palmityl (C₁₆), stearyl (C₁₈), and icosyl(C₂₀). In preferred embodiments, R¹ and R² are the same, i.e., R¹ and R²are both myristyl (i.e., dimyristyl), R¹ and R² are both stearyl (i.e.,distearyl), etc. Diacylglycerols have the following general formula:

The term “dialkyloxypropyl” refers to a compound having 2 alkyl chains,R¹ and R², both of which have independently between 2 and 30 carbons.The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, lauryl (C₁₂), myristyl (C₁₄), palmityl (C₁₆),stearyl (C₁₈), and icosyl (C₂₀). In preferred embodiments, R¹ and R² arethe same, i.e., R¹ and R² are both myristyl (i.e., dimyristyl), R¹ andR² are both stearyl (i.e., distearyl), etc.

In Formula VII above, the PEG has an average molecular weight rangingfrom about 550 daltons to about 10,000 daltons. In certain instances,the PEG has an average molecular weight of from about 500 daltons toabout 5,000 daltons (e.g., from about 1,000 daltons to about 5,000daltons, from about 1,500 daltons to about 3,000 daltons, from about 750daltons to about 3,000 daltons, from about 750 daltons to about 2,000daltons, etc.). In preferred embodiments, the PEG has an averagemolecular weight of about 2,000 daltons or about 750 daltons. The PEGcan be optionally substituted with alkyl, alkoxy, acyl, or aryl. Incertain embodiments, the terminal hydroxyl group is substituted with amethoxy or methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from.

In one embodiment, n is selected so that the resulting polymer chain hasa molecular weight of about 2000.

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a dilauryloxypropyl (C₁₂)—PEGconjugate, dimyristyloxypropyl (C₁₄)—PEG conjugate, adipalmityloxypropyl (C₁₆)—PEG conjugate, or a distearyloxypropyl(C₁₈)-PEG conjugate. Those of skill in the art will readily appreciatethat other dialkyloxypropyls can be used in the PEG-DAA conjugates ofthe present invention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

The lipid conjugate (e.g., PEG-lipid) typically comprises from about 0.1mol % to about 10 mol %, from about 0.5 mol % to about 10 mol %, fromabout 1 mol % to about 10 mol %, from about 0.6 mol % to about 1.9 mol%, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % toabout 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol %, fromabout 1 mol % to about 1.7 mol %, from about 1.2 mol % to about 1.8 mol%, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % toabout 1.6 mol %, or from about 1.4 mol % to about 1.5 mol % of the totallipid present in the particle.

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the nucleic acid-lipid particle is tobecome fusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe nucleic acid-lipid particle and, in turn, the rate at which thenucleic acid-lipid particle becomes fusogenic. For instance, when aPEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate isused as the lipid conjugate, the rate at which the nucleic acid-lipidparticle becomes fusogenic can be varied, for example, by varying theconcentration of the lipid conjugate, by varying the molecular weight ofthe PEG, or by varying the chain length and degree of saturation of theacyl chain groups on the phosphatidylethanolamine or the ceramide. Inaddition, other variables including, for example, pH, temperature, ionicstrength, etc. can be used to vary and/or control the rate at which thenucleic acid-lipid particle becomes fusogenic. Other methods which canbe used to control the rate at which the nucleic acid-lipid particlebecomes fusogenic will become apparent to those of skill in the art uponreading this disclosure.

Preparation of Lipid Particles

The lipid particles of the present invention, e.g., LNP, in which anactive agent or therapeutic agent such as a nucleic acid molecule isencapsulated in a lipid bilayer and is protected from degradation, canbe formed by any method known in the art including, but not limited to,a continuous mixing method or a direct dilution process.

In preferred embodiments, the cationic lipids are lipids of Formula I,II, and III, or combinations thereof. In other preferred embodiments,the non-cationic lipids are egg sphingomyelin (ESM),distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol, orcombinations thereof.

In certain embodiments, the present invention provides for LNP producedvia a continuous mixing method, e.g., a process that includes providingan aqueous solution comprising a nucleic acid, such as an interferingRNA or mRNA, in a first reservoir, providing an organic lipid solutionin a second reservoir, and mixing the aqueous solution with the organiclipid solution such that the organic lipid solution mixes with theaqueous solution so as to substantially instantaneously produce aliposome encapsulating the nucleic acid (e.g., interfering RNA or mRNA).This process and the apparatus for carrying this process are describedin detail in U.S. Patent Publication No. 20040142025, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a liposome substantially instantaneously upon mixing. As usedherein, the phrase “continuously diluting a lipid solution with a buffersolution” (and variations) generally means that the lipid solution isdiluted sufficiently rapidly in a hydration process with sufficientforce to effectuate vesicle generation. By mixing the aqueous solutioncomprising a nucleic acid with the organic lipid solution, the organiclipid solution undergoes a continuous stepwise dilution in the presenceof the buffer solution (i.e., aqueous solution) to produce a nucleicacid-lipid particle.

The LNP formed using the continuous mixing method typically have a sizeof from about 40 nm to about 150 nm, from about 50 nm to about 150 nm,from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, orfrom about 70 nm to about 90 nm. The particles thus formed do notaggregate and are optionally sized to achieve a uniform particle size.

In another embodiment, the present invention provides for LNP producedvia a direct dilution process that includes forming a liposome solutionand immediately and directly introducing the liposome solution into acollection vessel containing a controlled amount of dilution buffer. Inpreferred aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of liposomesolution introduced thereto. As a non-limiting example, a liposomesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides for LNPproduced via a direct dilution process in which a third reservoircontaining dilution buffer is fluidly coupled to a second mixing region.In this embodiment, the liposome solution formed in a first mixingregion is immediately and directly mixed with dilution buffer in thesecond mixing region. In preferred aspects, the second mixing regionincludes a T-connector arranged so that the liposome solution and thedilution buffer flows meet as opposing 1800 flows; however, connectorsproviding shallower angles can be used, e.g., from about 270 to about180°. A pump mechanism delivers a controllable flow of buffer to thesecond mixing region. In one aspect, the flow rate of dilution bufferprovided to the second mixing region is controlled to be substantiallyequal to the flow rate of liposome solution introduced thereto from thefirst mixing region. This embodiment advantageously allows for morecontrol of the flow of dilution buffer mixing with the liposome solutionin the second mixing region, and therefore also the concentration ofliposome solution in buffer throughout the second mixing process. Suchcontrol of the dilution buffer flow rate advantageously allows for smallparticle size formation at reduced concentrations.

These processes and the apparatuses for carrying out these directdilution processes are described in detail in U.S. Patent PublicationNo. 20070042031, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The LNP formed using the direct dilution process typically have a sizeof from about 40 nm to about 150 nm, from about 50 nm to about 150 nm,from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, orfrom about 70 nm to about 90 nm. The particles thus formed do notaggregate and are optionally sized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., LNP) can be sizedby any of the methods available for sizing liposomes. The sizing may beconducted in order to achieve a desired size range and relatively narrowdistribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids in the LNP are precondensed asdescribed in, e.g., U.S. patent application Ser. No. 09/744,103, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In other embodiments, the methods will further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brandname POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed LNP will range from about 0.01 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials also falls within this range.In other embodiments, the LNP preparation uses about 400 μg nucleic acidper 10 mg total lipid or a nucleic acid to lipid mass ratio of about0.01 to about 0.08 and, more preferably, about 0.04, which correspondsto 1.25 mg of total lipid per 50 μg of nucleic acid. In other preferredembodiments, the particle has a nucleic acid:lipid mass ratio of about0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed LNP will range from about 1 (1:1) to about 100(100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) toabout 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3(3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), fromabout 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1),from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25(25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) toabout 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5(5:1) to about 15 (15:1), from about 5 (5:1) to about 10 (10:1), about 5(5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9 (9:1), (10:1), 11 (11:1), 12 (12:1),13 (13:1), 14 (14:1), 15 (15:1), 16 (16:1), 17 (17:1), 18 (18:1), 19(19:1), 20 (20:1), 21 (21:1), 22 (22:1), 23 (23:1), 24 (24:1), 25(25:1), 26 (26:1), 27 (27:1), 28 (28:1), 29 (29:1) or 30 (30:1).

The ratio of the starting materials also typically falls within thisrange.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making LNP-CPLs (CPL-containing LNP)are discussed herein. Two general techniques include “post-insertion”technique, that is, insertion of a CPL into, for example, a pre-formedLNP, and the “standard” technique, wherein the CPL is included in thelipid mixture during, for example, the LNP formation steps. Thepost-insertion technique results in LNP having CPLs mainly in theexternal face of the LNP bilayer membrane, whereas standard techniquesprovide LNP having CPLs on both internal and external faces. The methodis especially useful for vesicles made from phospholipids (which cancontain cholesterol) and also for vesicles containing PEG-lipids (suchas PEG-DAAs and PEG-DAGs). Methods of making LNP-CPL, are taught, forexample, in U.S. Pat. Nos. 5,705,385; 6,586,410; 5,981,501; 6,534,484;and 6,852,334; U.S. Patent Publication No. 20020072121; and PCTPublication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Other methods for generating LNP may be found, for example, in U.S. Pat.No. 9,005,654 and PCT Publication No. WO 2007/012191, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

Kits

The present invention also provides lipid particles (e.g., LNP) in kitform. The kit may comprise a container which is compartmentalized forholding the various elements of the lipid particles (e.g., the activeagents or therapeutic agents such as nucleic acids and the individuallipid components of the particles). In some embodiments, the kit mayfurther comprise an endosomal membrane destabilizer (e.g., calciumions). The kit typically contains the lipid particle compositions of thepresent invention, preferably in dehydrated form, with instructions fortheir rehydration and administration.

As explained herein, the lipid particles of the invention (e.g., LNP)can be tailored to preferentially target particular tissues, organs, ortumors of interest. In certain instances, preferential targeting oflipid particles such as LNP may be carried out by controlling thecomposition of the particle itself. For instance, as set forth inExample 11, it has been found that the 1:57 PEG-cDSA LNP formulation canbe used to preferentially target tumors outside of the liver, whereasthe 1:57 PEG-cDMA LNP formulation can be used to preferentially targetthe liver (including liver tumors).

In certain other instances, it may be desirable to have a targetingmoiety attached to the surface of the lipid particle to further enhancethe targeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., LNP) are usefulfor the introduction of active agents or therapeutic agents (e.g.,nucleic acids, such as interfering RNA or mRNA) into cells. Accordingly,the present invention also provides methods for introducing an activeagent or therapeutic agent such as a nucleic acid (e.g., interfering RNAor mRNA) into a cell. The methods are carried out in vitro or in vivo byfirst forming the particles as described above and then contacting theparticles with the cells for a period of time sufficient for delivery ofthe active agent or therapeutic agent to the cells to occur.

The lipid particles of the invention (e.g., LNP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., LNP) can be administeredeither alone or in a mixture with a pharmaceutically-acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically-acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

The pharmaceutically-acceptable carrier is generally added followingparticle formation. Thus, after the particle is formed, the particle canbe diluted into pharmaceutically-acceptable carriers such as normalbuffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically-acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In Vivo Administration Systemic delivery for in vivo therapy, e.g.,delivery of a therapeutic nucleic acid to a distal target cell via bodysystems such as the circulation, has been achieved using nucleicacid-lipid particles such as those described in PCT Publication Nos. WO05/007196, WO 05/121348, WO 05/120152, and WO 04/002453, the disclosuresof which are herein incorporated by reference in their entirety for allpurposes. The present invention also provides fully encapsulated lipidparticles that protect the nucleic acid from nuclease degradation inserum, are nonimmunogenic, are small in size, and are suitable forrepeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA or mRNA) suspended indiluents such as water, saline, or PEG 400; (b) capsules, sachets, ortablets, each containing a predetermined amount of a therapeutic agentsuch as nucleic acid (e.g., interfering RNA or mRNA), as liquids,solids, granules, or gelatin; (c) suspensions in an appropriate liquid;and (d) suitable emulsions. Tablet forms can include one or more oflactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch,potato starch, microcrystalline cellulose, gelatin, colloidal silicondioxide, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA or mRNA)in a flavor, e.g., sucrose, as well as pastilles comprising thetherapeutic agent in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the therapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as LNP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, preferably between about 0.1and about 5 mg/kg of body weight, or about 108-10¹⁰ particles peradministration (e.g., injection).

In Vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA or mRNA) can be to any cell grownin culture, whether of plant or animal origin, vertebrate orinvertebrate, and of any tissue or type. In preferred embodiments, thecells are animal cells, more preferably mammalian cells, and mostpreferably human cells.

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the LNP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of LNP based ontheir relative effect on binding/uptake or fusion with/destabilizationof the endosomal membrane. This assay allows one to determinequantitatively how each component of the LNP or other lipid particleaffects delivery efficiency, thereby optimizing the LNP or other lipidparticle. Usually, an ERP assay measures expression of a reporterprotein (e.g., luciferase, (3-galactosidase, green fluorescent protein(GFP), etc.), and in some instances, a LNP formulation optimized for anexpression plasmid will also be appropriate for encapsulating aninterfering RNA or mRNA. In other instances, an ERP assay can be adaptedto measure downregulation of transcription or translation of a targetsequence in the presence or absence of an interfering RNA (e.g., siRNA).In other instances, an ERP assay can be adapted to measure theexpression of a target protein in the presence or absence of an mRNA. Bycomparing the ERPs for each of the various LNP or other lipid particles,one can readily determine the optimized system, e.g., the LNP or otherlipid particle that has the greatest uptake in the cell.

Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, e.g., hematopoietic precursor (stem) cells, fibroblasts,keratinocytes, hepatocytes, endothelial cells, skeletal and smoothmuscle cells, osteoblasts, neurons, quiescent lymphocytes, terminallydifferentiated cells, slow or noncycling primary cells, parenchymalcells, lymphoid cells, epithelial cells, bone cells, and the like. Inpreferred embodiments, an active agent or therapeutic agent such as oneor more nucleic acid molecules (e.g, an interfering RNA (e.g., siRNA) ormRNA) is delivered to cancer cells such as, e.g., lung cancer cells,colon cancer cells, rectal cancer cells, anal cancer cells, bile ductcancer cells, small intestine cancer cells, stomach (gastric) cancercells, esophageal cancer cells, gallbladder cancer cells, liver cancercells, pancreatic cancer cells, appendix cancer cells, breast cancercells, ovarian cancer cells, cervical cancer cells, prostate cancercells, renal cancer cells, cancer cells of the central nervous system,glioblastoma tumor cells, skin cancer cells, lymphoma cells,choriocarcinoma tumor cells, head and neck cancer cells, osteogenicsarcoma tumor cells, and blood cancer cells.

In vivo delivery of lipid particles such as LNP encapsulating one ormore nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) ormRNA) is suited for targeting cells of any cell type. The methods andcompositions can be employed with cells of a wide variety ofvertebrates, including mammals, such as, e.g, canines, felines, equines,bovines, ovines, caprines, rodents (e.g., mice, rats, and guinea pigs),lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans).

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,LNP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., LNP) are detectable in the subject at about 8, 12, 24,48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24,25, or 28 days after administration of the particles. The presence ofthe particles can be detected in the cells, tissues, or other biologicalsamples from the subject. The particles may be detected, e.g., by directdetection of the particles, detection of a therapeutic nucleic acid,such as an interfering RNA (e.g., siRNA) sequence or mRNA sequence,detection of the target sequence of interest (i.e., by detectingexpression or reduced expression of the sequence of interest), or acombination thereof.

Detection of Particles

Lipid particles of the invention such as LNP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA or mRNA) are detected andquantified herein by any of a number of means well-known to those ofskill in the art. The detection of nucleic acids may proceed bywell-known methods such as Southern analysis, Northern analysis, gelelectrophoresis, PCR, radiolabeling, scintillation counting, andaffinity chromatography. Additional analytic biochemical methods such asspectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), and hyperdiffusion chromatography may alsobe employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR) theligase chain reaction (LCR), Qβ-replicase amplification and other RNApolymerase mediated techniques (e.g., NASBA™) are found in Sambrook etal., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters, which can be changed or modified to yieldessentially the same results.

Examples

The present invention will be described in greater detail by way ofspecific examples. The following example is offered for illustrativepurposes, and is not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1. Synthesis of

(3-Chloropropyl)tris(decan-3-yloxy)silane (0.75 g, 1.21 mmol),dimethylamine (12.1 mmol (6.05 ml of 2.0 M in THF) and MeCN were heatedin a microwave in a sealed reactor vessel at 150° C. for ten minutes.After cooling, the reaction was partitioned between EtOAc and saturatedNaHCO₃. The organics were washed with water and brine, dried (Na₂SO₄)and concentrated in-vacuo. The residue was purified by automated columnchromatography (o-100% EtOAc/hexane) to giveN,N-dimethyl-3-(tris(decan-3-yloxy)silyl)propan-1-amine (0.16 g, 21.6%).1HNMR CDCl3 δ 3.85 (m, 3H), 2.23 (m, 8H), 1.5 (m 15H), 1.28 (m, 34H),0.87 (m, 19H), 0.55 (m, 2H).

The intermediate 3-Chloropropyl)tris(decan-3-yloxy)silane was preparedas follows.

a. Preparation of 3-Chloropropyl)tris(decan-3-yloxy)silane

Trichloro(3-chloropropyl)silane (1.72 g, 8.1 mmol) was stirred in Et₂Oat RT. A mixture of Undecan-3-ol (4.19 g, 24.3 mmol) and TEA (3.15 g,24.34 mmol) in Et₂O were added dropwise at RT. The reaction was stirredat RT for 16 hours. The resultant precipitate was removed by filtration,washing with additional Et₂O. The organics were washed sequentially withNaHCO₃, water and brine, dried (Na₂SO₄) and concentrated in-vacuo. Theresidue was purified by automated flash chromatography (2% EtOAc/hexane)to give (3-chloropropyl)tris(decan-3-yloxy)silane (2.48 g, 49.3%). 1HNMR CDCl3 δ 3.85 (p, 3H), 3.53 (t, 2H), 1.89 (m, 2H), 1.47 (m, 13H),1.28 (m, 35H), 0.9 (m, 18H), 0.7 (m, 2H)

Step 2: synthesis ofN,N-dimethyl-3-(tris(decan-3-yloxy)silyl)propan-1-amine (4) Examples2-23

Using a procedure similar to that described in Example 1, the followingcompounds (Examples 2-23) were prepared.

Example 2

Example 3

Example 4

Example 5

Example 6

Example 7

Example 8

Example 9

Example 10

Example 11

Example 12

Example 13

Example 14

Example 15

Example 16

Example 17

Example 18

Example 19

Example 20

Example 21

Example 22

Example 23

Example 24 Preparation of Lipid Formulations

Lipid stocks were prepared (about 7 mg/mL total lipid content) in 100%ethanol, using the lipid identities and molar ratios described. The mRNAwas diluted in acetate pH 5 and nuclease-free water to reach aconcentration of 0.366 mg/mL mRNA in 100 mM acetate pH 5. Equal volumesof each solution were blended at 400 mL/min in a T-connector, anddiluted with about 4 volumes of PBS, pH 7.4, using the direct dilutionmethod described in U.S. Pat. No. 9,404,127. Formulations were thenplaced in Slide-A-Lyzer dialysis units (MWCO 10,000) and were dialyzedovernight 10 mM Tris, 500 mM NaCl pH 8 (Tris/NaCl buffer). Followingdialysis the formulations were concentrated to about 0.6 mg/mL usingVivaSpin concentrator units (MWCO 100,000) and then filtered through a0.2 um syringe filter.

Example 24a

The various lipids of the present invention were formulated in LNPcompositions with the following molar ratios: DSPC (11%):Chol(33%):PEG₂₀₀₀-C-DMA (1.6%):Lipid X (55%). A composition was generatedcomprising the following components:

(a) nucleic acid;(b) a mixture of cholesterol and DSPC;(c) a conjugated lipid of formula:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; and(d) a cationic lipid of formula:

wherein the conjugated lipid comprised about 1.5 mol % of the totallipid present in the particle; DSPC comprised about 10 mol % of thetotal lipid present in the particle; cholesterol comprised about 38.5mol % of the total lipid present in the particle; and the cationic lipidcomprised about 50 mol % of the total lipid present in the particle; and

wherein the lipid to nucleic acid ratio was about 19.6.

Example 24b

A composition was generated comprising the following components:

(a) nucleic acid;(b) a mixture of cholesterol and DSPC;(c) a conjugated lipid of formula:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; and(d) a cationic lipid of formula:

wherein the conjugated lipid comprised about 1.6 mol % of the totallipid present in the particle; DSPC comprised about 11 mol % of thetotal lipid present in the particle; cholesterol comprised about 33 mol% of the total lipid present in the particle; and the cationic lipidcomprised about 55 mol % of the total lipid present in the particle; and

wherein the lipid to nucleic acid ratio was about 20.5.

Example 24c

A composition was generated comprising the following components:

(a) nucleic acid;(b) a mixture of cholesterol and DSPC;(c) a conjugated lipid of formula:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; and(d) a cationic lipid of formula:

wherein the conjugated lipid comprised about 1.6 mol % of the totallipid present in the particle; DSPC comprised about 10.9 mol % of thetotal lipid present in the particle; cholesterol comprised about 32.8mol % of the total lipid present in the particle; and the cationic lipidcomprised about 54.9 mol % of the total lipid present in the particle;and

wherein the lipid to nucleic acid ratio was about 20.2.

Example 25 In Vivo Assay

Generally, the LNP were injected intravenously at 0.5 mg/kg to femaleBalb/C mice, 5-8 weeks old and blood was collected at 4-6 hours postdosing; blood is collected into K2EDTA and processed to plasma, thenstored frozen at −80° C. until analysis. Activity was assayed by testingthe plasma for human EPO expression using an human EPO ELISA kit eitherfrom StemCell (catalogue #01630) or R&D Systems (catalogue DEP00)following the manufacturer's instructions. Data is provided in thefollowing Table.

As can be seen from the data in Table 1, some of the compositions areconsiderably more potent than the MC3 composition used in patisiran(Onpattro), an approved LNP product for treatment of TTR amyloidosis.

TABLE 1 Efficacy of 0.5 mg/kg LNP containing the cationic lipids of thepresent invention (at 55 mol %, with 1.6 mol % PEG2000-C-DMA, 33%cholesterol and 11% DSPC), and human EPO mRNA, 4 h Following IV Dosingin Balb/C Mice (n = 4-5) Composition EPO Stdev (Lipid X =) (mU/mL)(mU/mL) Example 1  60445 5608 Example 2  144176 42954 Example 4  130080748843 Example 6  25596 955 Example 7  942452 290341 Example 8  304462024 Example 9  37927 1949 Example 11 256974 35579 Example 13 31063352332 Example 15 18385 756 Example 17 226593 42270 Example 18 1037188661 Example 19 47218 3408 Example 20 2242992 228147 Example 21 14704192130352 Example 22 15075 8524 Example 23 17488 400 Patisiran Composition42230 2697 with MC3 (1.5:50.0:38.5:10.0)

1. A nucleic acid-lipid particle comprising: (a) one or more nucleicacid molecules: (b) a non-cationic lipid; (c) a conjugated lipid; and(b) a cationic lipid of formula (I):

wherein: R¹ is a C₂-C₃₀ hydrocarbyl; R² is a C₂-C₃₀ hydrocarbyl; R³ is aC₂-C₃₀ hydrocarbyl, X is a divalent C₂-C₈ alkyl; R⁴ is NR^(a)R^(b); andeach R^(a) and R^(b) is independently selected from the group consistingof methyl, ethyl, propyl, cyclopropyl, and butyl, which methyl, ethyl,propyl, cyclopropyl, and butyl is optionally substituted with hydroxy;or R^(a) and R^(b) taken with the nitrogen to which they are attachedform an aziridine, azetidine, proline, piperidine, piperazine, ormorpholine ring, which ring is optionally substituted with hydroxyl orwith C₁-C₆ alkyl that is optionally substituted with hydroxy; andwherein the one or more nucleic acid molecules are encapsulated withinthe lipid particle. 2-5. (canceled)
 6. The nucleic acid-lipid particleof claim 1, wherein R¹, R² and R³ are each independently a(C₂-C₂₀)alkyl, (C₂-C₂₀)alkenyl, or (C₂-C₂₀)alkynyl. 7-9. (canceled) 10.The nucleic acid-lipid particle of claim 1, wherein R¹, R² and R³ areeach independently a (C₈-C₂₀)alkenyl, having only one double bond.11-36. (canceled)
 37. The nucleic acid-lipid particle of claim 1,wherein each R^(a) and R^(b) is independently selected from the groupconsisting of methyl, ethyl, propyl, cyclopropyl, and butyl, whichmethyl, ethyl, propyl, cyclopropyl, and butyl is optionally substitutedwith hydroxy.
 38. The nucleic acid-lipid particle of claim 1, whereinR^(a) and R^(b) taken with the nitrogen to which they are attached forman aziridine, azetidine, proline, piperidine, piperazine, or morpholinering, which ring is optionally substituted with hydroxyl or with C₁-C₆alkyl that is optionally substituted with hydroxy.
 39. The nucleicacid-lipid particle of claim 1, wherein each R^(a) and R^(b) isindependently selected from the group consisting of methyl and ethyl.40. The nucleic acid-lipid particle of claim 1, wherein R⁴ isdimethylamino. 41-42. (canceled)
 43. The nucleic acid-lipid particle ofclaim 1, wherein the nucleic acid is selected from the group consistingof small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpinRNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA),mRNA, tRNA, rRNA, viral RNA (vRNA), self-amplifying RNA, andcombinations thereof.
 44. The nucleic acid-lipid particle of claim 43,wherein the nucleic acid is an mRNA molecule.
 45. The nucleic acid-lipidparticle of claim 43, wherein the nucleic acid comprises a doublestranded siRNA molecule.
 46. The nucleic acid-lipid particle of claim45, wherein the double stranded siRNA molecule comprises at least onemodified nucleotide. 47-54. (canceled)
 55. The nucleic acid-lipidparticle of claim 1, wherein the conjugated lipid comprises apolyethyleneglycol (PEG)-lipid conjugate. 56-80. (canceled)
 81. Apharmaceutical composition comprising a nucleic acid-lipid particle ofclaim 1, and a pharmaceutically acceptable carrier.
 82. A method forintroducing a nucleic acid into a cell, the method comprising:contacting the cell with a nucleic acid-lipid particle of claim
 1. 83.(canceled)
 84. A method for the in vivo delivery of a nucleic acid, themethod comprising: administering to a mammalian subject a nucleicacid-lipid particle of claim
 1. 85-87. (canceled)
 88. A method fortreating a disease or disorder in a mammalian subject in need thereof,the method comprising: administering to the mammalian subject atherapeutically effective amount of a nucleic acid-lipid particle ofclaim
 1. 89. The method of claim 88, wherein the disease or disorder isselected from the group consisting of a viral infection, a liver diseaseor disorder, and cancer. 90-95. (canceled)
 96. A method for preparing acompound of formula (I):

as described in claim 1, comprising reacting a corresponding compound offormula (Ia):

wherein R^(4a) is bromo or chloro, with an amine of formula HNR^(a)R^(b)to provide the compound of formula (I) wherein R⁴ is NR^(a)R^(b). 97.(canceled)
 98. A compound of formula (I):

wherein R¹ is a C₂-C₃₀ hydrocarbyl; R² is a C₂-C₃₀ hydrocarbyl; R³ is aC₂-C₃₀ hydrocarbyl; X is a divalent C₂-C₈ alkyl; R⁴ is NR^(a)R^(b); eachR^(a) and R^(b) is independently selected from the group consisting ofmethyl, ethyl, propyl, cyclopropyl, and butyl, which methyl, ethyl,propyl, cyclopropyl, and butyl is optionally substituted with hydroxy;or R and R^(b) taken with the nitrogen to which they are attached forman aziridine, azetidine, proline, piperidine, piperazine, or morpholinering, which ring is optionally substituted with hydroxyl or with C₁-C₆alkyl that is optionally substituted with hydroxy.
 99. (canceled) 100.The nucleic acid-lipid particle of claim 1, wherein the cationic lipidof formula (I) is a lipid of the formula: